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Cobb ross cell signaling chapter

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Nusrat Gulbarga
Nusrat Gulbarga

Cell Signaling is an important facet of biological life. It allows cells to perceive and respond to the extracellular environment allowing development, growth, immunity, etc. Additionally, errors in cell signaling may result in cancer growth, diabetes. ... The inducer does not diffuse from the cell producing it.

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nucleus lipid droplet Golgi RasGAP Ser338 Tyr341 Ser4 Ser62 Raf Tyr Gab2 PI3K Ser Thr SOS Grb2 Grb2 Ser Thr SOSGrb2 Ser Thr SOS Grb2 Ser Thr SOSGrb2 Ser Thr SOS GTP GDP GDP Ras GDP Ras GTP Ras GTP Ras RasGAP P Tyr Gab2 Tyr580 Tyr542 SHP-2 P Tyr Gab2 Tyr580Tyr542SHP-2 P Tyr Gab2 Tyr580Tyr542SHP-2 Tyr544 Tyr559 Tyr697 Tyr706 Tyr721 Tyr807 Tyr921Tyr974 M-CSFR Tyr706 P P Tyr706 P P Tyr701 Ser727STAT1 P Tyr701 P Ser727STAT1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 Tyr Ser312(307:R) IRS Tyr IL-4R Tyr JAK1 TyrJAK3 Tyr JAK1 Tyr IL-4R Tyr JAK1 Tyr IL-4R Tyr common chain TyrJAK3 Tyr common chain TyrJAK3 Tyr common chain y yy y P y P yJ 3 P y P yJ 3 SOCS1 /JAB P Tyr Ser312(307:R) IRS TyrSTAT6 P Tyr STAT6 P Tyr P Tyr STAT6 P Tyr P Tyr STAT6 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 Tyr Tyr STAT6 Tyr440 IFN R1 Tyr JAK1 Tyr1007 JAK2 Tyr JAK1 Tyr440 IFN R1 Tyr JAK1 Tyr440 IFN R1 SHP-1 SOCS1/JAB Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R IL-6R gp130 Tyr JAK1 TyrTyk2 Tyr JAK1 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R Tyr JAK1 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R TyrTyk2 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R TyrTyk2 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R Tyr JAK1 TyrTyk2Tyr JAK1 TyrTyk2 SOCS3 SHP-1 Tyr580 Tyr542 SHP-2 P Tyr580 P Tyr542 SHP-2 Tyr STAT3 P Tyr STAT3 P Tyr P Tyr STAT3 P Tyr P Tyr STAT3 SOCS3 TyrTyk2Tyr IL-10R IL-10RIL-10R Tyr JAK1 Tyr JAK1 Tyr IL-10R Tyr JAK1 Tyr IL-10R TyrTyk2 Tyr IL-10R TyrTyk2 Tyr IL-10R P PP P TLR4 MD-2 IKK TBK-1 P Ser386 P Ser385 IRF-3 Ser Thr Lys IRAK1 IRAK-M SOCS1 /JAB P Ser P Thr Lys IRAK1 Ubc13 Uev1A Ubc13 Uev1A TRIF/ TICAM-1 TBK-1 IKK Ser386 Ser385 IRF-3 IFN- CAPK IKK Ser176 Ser181 IKK IKK IKK Ser176 Ser181 IKK IKK IKK P Ser176 P Ser181 IKK IKK IKK P Ser176 P Ser181 IKK IKK CAPK SCF TrCP P Thr183 P Tyr185 ERK1 ERK2 P Ser276 Ser529 NF- B p65+p50 P Ser276 P Ser529 NF- B p65+p50 Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B IL-10 IL-6 IL-1 p50 I B TNFTNF P Thr183 P Tyr185 P Thr183 P Tyr185 ERK1 ERK2 Ser338 Tyr341 Ser4 Ser62 Raf Tyr STAT5 GM-CSFR GM-CSFR Tyr GM-CSFR TyrJAK1 Tyr1007 JAK2 P Tyr STAT5 P Tyr P Tyr STAT5 P Tyr P Tyr STAT5 Tyr701 Ser727STAT1 IRF-2 IRF-1 IRF-9 Ser484 Ser485 IRF-7 P Tyr701 P Ser727STAT1 P Tyr STAT2 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 IRF-1IRF-2 IFN- IRF-1 IRF-2 NOSII/iNOS IRF-7 IRF-9 P Ser484 P Ser485 IRF-7 P Ser484 P Ser485 IRF-7 IFN- IFN- pyruvate pyruvate acetyl CoA NAD+ NADH+H+ pyruvate carboxylase pyruvate dehydrogenase PDH kinase pyruvate carrier P P P pyruvate dehydrogenase PDH kinase citrate acyl-CoA carnitine CoASH acylcarnitine fatty acid malonyl CoA carnitine acylcarnitine acyl-CoA CPT I CPT II CACT CoASH acyl CoA synthetase fatty acid synthetase acetyl CoA oxaloacetate CoASH acetyl CoA carboxylase citrate liase malate malate dehydrogenase malic enzyme glycerol 3P TG TG O2 hypoxanthine NADPH xanthine e- NADP+ F H2O2 Cl- LOOH Fe3+ e- .O 2 - HOCl acyl CoA synthetase 27-hydroxyChol LXR 9 r 27-hydroxyChol LXR 9 r LXR LXRLXR R SCAP Site Site-2 protease ? acetyl CoA carboxylase fatty acid synthetase Ser383 Ser389Elk-1 P Ser383 P Ser389 Elk-1 PKA Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B PKA PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 PKA Ser276 Ser529 NF- B p65+p50 PKA CK II NOSII/iNOS NADPH oxidase xanthine oxidase SOD MPO ATP synthetase ATP ADP O2 H+ e- H+ PP2A ys63TRAF6 SerThr LysIRAK1 ys63TRAF6 SerThr LysIRAK1 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 TAB2 Ser ThrTAB1Ser192Thr184 Thr187TAK1 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 P Ser P Thr Ub LysIRAK1 P Ser P ThrTAB1 P Ser192 P Thr184 P Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P ThrTAB1 P Ser192 P Thr184 P Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 c-fos c-jun AP-1 c-Fos+c-Jun Ser21 Ser32 Ser42 Ser70 Ser113 Ser374 c-Fos Ser63 Ser73 c-Jun P Ser63 P Ser73 c-Jun P Ser21P Ser32 P Ser42 P Ser70 P Ser113 P Ser374 c-Fos P Ser63 P Ser73 c-Jun p53 IL-4 IL-4 0 TRAF2 TRAF1 A20TRAF2 TRAF1 A20 IFN IFN A20 PAFR calpain TRAM TRAMTRAM Tyr580 Tyr542 SHP-2 IL-1ra IL-1ra P Ser386 P Ser385 P Ser386 P Ser385 IRF-3 P Ser386 P Ser385 P Ser386 P Ser385 IRF-3 P Tyr STAT6 P Ser276 P Ser529 P Ser276 P Ser529 NF- B p65+p50 P Ser276 P Ser529 P Ser276 P Ser529 NF- B p65+p50 GM-CSF GM-CSF Tyr STAT3 P Tyr STAT3 -2 P Tyr580 P Tyr542 SHP-2 K PI3K Tyr STAT5 P Tyr STAT5 P Tyr P Tyry P Tyr P Tyry P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr JAK1 P TyrTyk2 SOCS3 P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr JAK1 P TyrTyk2 SOCS3 Tyr IFN R2 P Tyr440 IFN R1 P Tyr IFN R2 P Tyr JAK1 P Tyr1007 JAK2 SOCS1 /JAB P Tyr440 IFN R1 P Tyr IFN R2 P Tyr JAK1 P Tyr1007 JAK2 SOCS1 /JAB Tyr1007 JAK2 Tyr IFN R2 Tyr1007 JAK2 Tyr IFN R2 y Tyr1007y Tyr1007 P y P Tyr1007 P y P Tyr1007 IL-4R p38MAPK P Ser473 P Thr38 Akt/PKB IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 PIAS3 PIAS3 P Tyr P Tyr STAT3 PIAS3 P Tyr P Tyr STAT3 Tyr701 Ser727 Tyr701 Ser727STAT1 MKP PIAS1 PIAS1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 PIAS1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 P TyrJAK1 P TyrJAK3 P Tyr IL-4R P Tyr common chain SOCS1 /JAB P TyrJAK1 P TyrJAK3 P Tyr IL-4R P Tyr common chain SOCS1 /JAB SHP-1 SHIP Tyr Fyn P Tyr Fyn PI3K PI3K Ser473Thr38 Akt/PKB P Tyr P Thr JNK proteasome P Ser21P Ser32 P Ser42 P Ser70 P Ser113 P Ser374 c-Fos PP2B Thr183 Tyr185 Thr183 Tyr185 ERK1 ERK2 MKP LXRRXR CPT1 SREBP1c / bHLH SREBP1c / bHLH SREBP1c / bHLH Tyr chain Tyr Tyr chain Fc RIa chain Tyr518 Tyr519 Syk Tyr518 Tyr519 Syk P Tyr518 P Tyr519 Syk P Tyr771 P Tyr783 P Tyr1254 PLC Tyr771 Tyr783 Tyr1254 PLC PP2APP2B Pi P Tyr580 P Tyr542SHP-2 P Tyr580 P Tyr542 SHP-2 P Tyr Gab2 P Tyr580 P Tyr542SHP-2 P Tyr Gab2 P Tyr580 P Tyr542SHP-2 P Ser63 P Ser73 P Ser63 P Ser73 c-Jun P Ser369 P Thr577 P Ser386 P Ser227RSK P Ser369 P Thr577 P Ser386 Ser227 RSK Ser133 CREB P Ser133 CREB Grb2 P Ser P Thr SOSGrb2 P Ser P Thr SOS ASK P MEKK SEK1/MKK4 SEK2/MKK7 P SEK1/MKK4 P SEK2/MKK7 P TyrThr JNK TyrThr JNK Ser473Thr38 Akt/PKB P Ser473 P Thr38 Akt/PKB Tyr P Ser312(307:R) IRS SOCS3 P Tyr P Thr JNK P Thr183 P Tyr185 ERK1 ERK2 P Ser369 P Ser386 R Src P UbcH5 p50 p60 TICAM-1TICAM-1 TRIF/ TICAM-1 CHAPTER OUTLINE 589 Melanie H. Cobb and Elliott M. Ross The University of Texas Southwestern Medical Center at Dallas Introduction Cellular signaling is primarily chemical Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Receptors are catalysts and amplifiers Ligand binding changes receptor conformation Signals are sorted and integrated in signaling pathways and networks Cellular signaling pathways can be thought of as biochemical logic circuits Scaffolds increase signaling efficiency and enhance spatial organization of signaling Independent, modular domains specify protein-protein interactions Cellular signaling is remarkably adaptive Signaling proteins are frequently expressed as multiple species Activating and deactivating reactions are separate and independently controlled Cellular signaling uses both allostery and covalent modification Second messengers provide readily diffusible pathways for information transfer Ca2+ signaling serves diverse purposes in all eukaryotic cells Lipids and lipid-derived compounds are signaling molecules PI 3-kinase regulates both cell shape and the activation of essential growth and metabolic functions Signaling through ion channel receptors is very fast Nuclear receptors regulate transcription G protein signaling modules are widely used and highly adaptable Heterotrimeric G proteins regulate a wide variety of effectors Heterotrimeric G proteins are controlled by a regulatory GTPase cycle Small, monomeric GTP-binding proteins are multiuse switches Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell Two-component protein phosphorylation systems are signaling relays Pharmacological inhibitors of protein kinases may be used to understand and treat disease Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function The Wnt pathway regulates cell fate during development and other processes in the adult Diverse signaling mechanisms are regulated by protein tyrosine kinases Src family protein kinases cooperate with receptor protein tyrosine kinases MAPKs are central to many signaling pathways Cyclin-dependent protein kinases control the cell cycle Diverse receptors recruit protein tyrosine kinases to the plasma membrane What’s next? Summary References 14.36 14.35 14.34 14.33 14.32 14.31 14.30 14.29 14.28 14.27 14.26 14.25 14.24 14.23 14.22 14.21 14.20 14.19 14.18 14.17 14.16 14.15 14.14 14.13 14.12 14.11 14.10 14.9 14.8 14.7 14.6 14.5 14.4 14.3 14.2 14.1 Principles of cell signaling 14 This image represents about 10% of the map of the known signaling interactions and reactions in the mouse macrophage. Preparing such a map in a computable format is the first step in analyzing a large signaling network. This map was prepared by the group led by Hiroaki Kitano at the Systems Biology Institute, Tokyo, using their CellDesigner program. Map courtesy of Kanae Oda, Yukiko Matsuoka, and Hiroaki Kitano (The Systems Biology Institute). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 589
590 CHAPTER 14 Principles of cell signaling nearby), odors, molecules that regulate growth or differentiation, and proteins on the outside of adjacent cells. A mammalian cell typically expresses about fifty distinct receptors that sense different inputs, and, overall, mammals express several thousand receptors. Despite the diversity of cellular lifestyles and the enormous number of substances sensed by different cells, the general classes of proteins and mechanisms involved in signal transduc- tion are conserved throughout living cells, as shown in FIGURE 14.1. • G protein-coupled receptors, composed of seven membrane-span- ning helices, promote activation of het- erotrimeric GTP-binding proteins called G proteins, which associate with the in- ner face of the plasma membrane and convey signals to multiple intracellular proteins. • Receptor protein kinases are often dimers of single membrane-spanning proteins that phosphorylate their in- tracellular substrates and, thus, change the shape and function of the target pro- teins. These protein kinases frequently contain protein interaction domains that organize complexes of signaling pro- teins on the inner surface of the plasma membrane. • Phosphoprotein phosphatases re- verse the effect of protein kinases by re- moving the phosphoryl groups added by protein kinases. • Other single membrane-spanning en- zymes, such as guanylyl cyclase, have an overall architecture similar to the re- ceptor protein kinases but different en- zymatic activities. Guanylyl cyclase catalyzes the conversion of GTP to 3′:5′- cyclic GMP, which is used to propagate the signal. • Ion channel receptors, although di- verse in detailed structure, are usually oligomers of subunits that each contain several membrane-spanning segments. The subunits change their conforma- tions and relative orientations to per- mit ion flux through a central pore. • Two-component systems may either be membrane spanning or cytosolic. The number of their subunits is also vari- able, but each two-component system contains a histidine kinase domain or subunit that is regulated by a signaling molecule and a response regulator that Introduction All cells, from prokaryotes through plants and animals, sense and react to stimuli in their en- vironments with stereotyped responses that al- low them to survive, adapt, and function in ways appropriate to the needs of the organism. These responses are not simply direct physical or metabolic consequences of changes in the local environment. Rather, cells express arrays of sensing proteins, or receptors, that recognize specific extracellular stimuli. In response to these stimuli, receptors regulate the activities of diverse intracellular regulatory proteins that in turn initiate appropriate responses by the cell. The process of sensing external stimuli and conveying the inherent information to intra- cellular targets is referred to as cellular signal transduction. Cells respond to all sorts of stimuli. Microbes respond to nutrients, toxins, heat, light, and chemical signals secreted by other microbes. Cells in multicellular organisms express recep- tors specific for hormones, neurotransmitters, autocrine and paracrine agents (hormone- like compounds from the secreting cell or cells 14.1 Response regulator Sensor Histidine kinase( ( E1 E2 E1 E2 Hetero- trimeric G protein (GPCR) G protein coupled receptor Trans- membrane scaffold Guanylyl cyclaseReceptor protein kinase Ion channel Two- component complex Transcription factor NUCLEUS Overview of major receptor types in a cell FIGURE 14.1 Receptors form a rather small number of families that share com- mon mechanisms of action and overall similar structures. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 590
14.2 Cellular signaling is primarily chemical 591 contains a phosphorylatable aspartate (Asp) residue. • Some receptors are transmembrane scaffolds that change either the con- formation or oligomerization of their intracellular scaffold domains in re- sponse to extracellular signaling mole- cules, or ligands, and, thus, recruit interacting regulatory proteins to a com- mon site on the membrane. • Nuclear receptors are transcription factors, often heterodimers, that may reside in the cytoplasm until activated by agonists or may be permanently lo- cated in the nucleus. The biochemical processes of signal trans- duction are strikingly similar among cells. Bacteria, fungi, plants, and animals use similar proteins and multiprotein modules to detect and process signals. For example, evolutionar- ily conserved heterotrimeric G proteins and G protein-coupled receptors are found in plants, fungi, and animals. Similarly, 3′:5′ cyclic AMP (cAMP) is an intracellular signaling molecule in bacteria, fungi, and animals; and Ca2+ serves a similar role in all eukaryotes. Protein kinases and phosphoprotein phosphatases are used to regulate enzymes in all cells. Although the basic biochemical components and processes of signal transduction are con- served and reused, they are often used in wildly divergent patterns and for many different phys- iological purposes. For example, cAMP is synthe- sized by distantly related enzymes in bacteria, fungi, and animals, and acts on different pro- teins in each organism; it is a pheromone in some slime molds. Cells often use the same series of signaling proteins to regulate a given process, such as transcription, ion transport, locomotion, and metabolism. Such signaling pathways are as- sembled into signaling networks to allow the cell to coordinate its responses to multiple in- puts with its ongoing functions. It is now pos- sible to discern conserved reaction sequences in and between pathways in signaling networks that are analogous to devices within the circuits of analog computers: amplifiers, logic gates, feedback and feed-forward controls, and mem- ory. This chapter discusses the principles and strategies of cellular signaling first and then dis- cusses the conserved biochemical components and reactions of signaling pathways and how these principles are applied. Cellular signaling is primarily chemical Most signals sensed by cells are chemical, and, when physical signals are sensed, they are gen- erally detected as chemical changes at the level of the receptor. For example, the visual pho- toreceptor rhodopsin is composed of the pro- tein opsin, which binds to a second component, the colored vitamin A derivative cis-retinal (the chromophore). When cis-retinal absorbs a photon, it photoisomerizes to trans-retinal, which is an activating ligand of the opsin pro- tein. (For more on rhodopsin signaling see 14.20 G protein signaling modules are widely used and highly adaptable). Similarly, plants sense red and blue light using the photosensory proteins phy- tochrome and cryptochrome, which detect pho- tons that are absorbed by their tetrapyrrole or flavin chromophores. Cryptochrome homologs are also expressed in animals, where they prob- ably mediate adjustment of the diurnal cycle. A few receptors do respond directly to phys- ical inputs. Pressure-sensing channels, which ex- ist in one form or another in all organisms, mediate responses to pressure or shear by chang- ing their ionic conductance. In mammals, hear- ing is mediated indirectly by a mechanically operated channel in the hair cell of the inner ear. The extracellular domain of a protein called cad- herin is pulled in response to acoustic vibration, generating the force that opens the channel. Cells sense mechanical strain through a number of cell surface proteins, including inte- grins. Integrins provide signals to cells based on their attachment to other cells and to molecu- lar complexes in the external milieu. One major group of physically responsive receptors is made up of channels that sense elec- tric fields. Another interesting group are heat/pain-sensing ion channels; several of these heat-sensitive ion channels also respond to chemical compounds, such as capsaicin, the “hot” lipid irritant in hot peppers. Whether a signal is physical or chemical, the receptor initiates the reactions that change the behavior of the cell. We will discuss how these effects are generated in the rest of the chapter. Key concepts • Cells can detect both chemical and physical signals. • Physical signals are generally converted to chemical signals at the level of the receptor. 14.2 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 591
592 CHAPTER 14 Principles of cell signaling Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Receptors mediate responses to amazingly di- verse extracellular messenger molecules; hence, the cell must express a large number of recep- tor varieties, each able to bind its extracellular ligand. In addition, each receptor must be able to initiate a cellular response. Receptors, thus, contain two functional domains: a ligand- binding domain and an effector domain, which may or may not correspond to definable structural domains within the protein. The separation of ligand-binding and effec- tor functions allows receptors for diverse ligands to produce a limited number of evolutionarily conserved intracellular signals through the ac- tion of a few effector domains. In fact, there are Key concepts • Receptors contain a ligand-binding domain and an effector domain. • Receptor modularity allows a wide variety of signals to use a limited number of regulatory mechanisms. • Cells may express different receptors for the same ligand. • The same ligand may have different effects on the cell depending on the effector domain of its receptor. 14.3 only a limited number of receptor families, which are related by their conserved structures and sig- naling functions (see Figure 14.1). There are several useful correlates to the two-domain nature of receptors. For example, a cell can control its responsiveness to an extra- cellular signal by regulating the synthesis or degradation of a receptor or by regulating the receptor’s activity (see 14.10 Cellular signaling is remarkably adaptive). In addition, the nature of a response is gen- erally determined by the receptor and its effec- tor domain rather than any physicochemical property of the ligand. FIGURE 14.2 illustrates the concept that a ligand may bind to more than one kind of receptor and elicit more than one type of response, or several different ligands may all act identically by binding to function- ally similar receptors. For example, the neuro- transmitter acetylcholine binds to two classes of receptors. Members of one class are ion chan- nels; members of the other regulate G proteins. Similarly, steroid hormones bind both to nu- clear receptors, which bind chromatin and reg- ulate transcription, and to other receptors in the plasma membrane. Conversely, when multiple ligands bind to receptors of the same biochemical class, they generate similar intracellular responses. For ex- ample, it is not uncommon for a cell to express several distinct receptors that stimulate produc- tion of the intracellular signaling molecule cAMP. The effect of the receptor on the cell will also be determined significantly by the biology of the cell and its state at any given time. Ligand binding and effector domains may evolve independently in response to varied se- lective pressures. For example, mammalian and invertebrate rhodopsins transduce their signal through different effector G proteins (Gt and Gq, respectively). Another example is calmod- ulin, a small calcium-binding regulatory pro- tein in animals, which in plants appears as a distinct domain in larger proteins. The receptor’s two-domain nature allows the cell to regulate the binding of ligand and the effect of ligand independently. Covalent modification or allosteric regulation can al- ter ligand-binding affinity, the ability of the lig- and-bound receptor to generate its signal or both. We will discuss these concepts further in 14.13 Cellular signaling uses both allostery and co- valent modification. Receptors can be classified either accord- ing to the ligands they bind or the way in which they signal. Signal output, which is character- Ligand A Ligand A Output 1 Output 2 Output 2 Output 1 Output 1 Ligand B Ligand C LBD1 LBD1 LBD1 LBD2 LBD3 ED1 ED1 ED1ED2 ED2 Receptors have a ligand-binding domain and an effector domain CHIMERIC RECEPTOR FIGURE 14.2 Receptors can be thought of as composed of two functional do- mains, a ligand-binding domain (LBD) and an effector domain (ED). The two- domain property implies that two receptors that respond to different ligands (middle) could initiate the same function by activating similar effector do- mains, or that a cell could express two receptor isoforms (left) that respond to the same ligand with distinct cellular effects mediated by different effector do- mains. It also implies that one can create an artificial chimeric receptor with novel properties. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 592
14.5 Ligand binding changes receptor conformation 593 istic of the effector domain, usually correlates best with overall structure and sequence con- servation. (Receptor families grouped by their functions are the organizational basis of the sec- ond half of this chapter.) However, classifying receptors pharmacologically, according to their specificity for ligands, is particularly useful for understanding the organization of endocrine and neuronal systems and for categorizing the multiple physiological responses to drugs. Expression of a receptor that is not nor- mally expressed in a cell is often sufficient to confer responsiveness to that receptor’s ligand. This responsiveness often occurs because the cell expresses the other components necessary for propagating the intracellular signal from the receptor. The precise nature of the response will reflect the biology of the cell. Experimentally, responsiveness to a compound can be induced by introducing the cDNA that encodes the re- ceptor. For example, mammalian receptors may be expressed in yeast, such that the yeast re- spond visibly to receptor ligands, thus provid- ing a way to screen for new chemicals (drugs) that activate the receptor. Finally, it is possible to create chimeric re- ceptors by fusing the ligand-binding domain from one receptor with the effector domain from a different receptor (Figure 14.2). Such chimeras can mediate novel responses to the ligand. With genetic modification of the ligand- binding domain, receptors can be reengineered to respond to novel ligands. Thus, scientists can manipulate cell functions with nonbiological compounds. Receptors are catalysts and amplifiers Receptors act to accelerate intracellular func- tions and are, thus, functionally analogous to en- zymes or other catalysts. Some receptors, including the protein kinases, protein phos- phatases, and guanylate cyclases, are themselves enzymes and thus classical biochemical cata- lysts. More generally, however, receptors use the relatively small energy of ligand binding to accelerate reactions that are driven by alterna- tive energy sources. For example, receptors that are ion channels catalyze the movement of ions Key concepts • Receptors act by increasing the rates of key regulatory reactions. • Receptors act as molecular amplifiers. 14.4 across membranes, a process driven by the elec- trochemical potential developed by distinct ion pumps. G protein-coupled receptors and other guanine nucleotide exchange factors catalyze the exchange of GDP for GTP on the G protein, an energetically favored process dictated by the cell’s nucleotide energy balance. Transcription factors accelerate the formation of the transcrip- tional initiation complex, but transcription it- self is energetically driven by multiple steps of ATP and dNTP hydrolysis. As catalysts, receptors enhance the rates of reactions. Most signaling involves kinetic rather than thermodynamic regulation; that is, sig- naling events change reaction rates rather than their equilibria (see the next section). Thus, sig- naling is similar to metabolic regulation, in which specific reactions are chosen according to their rates, with thermodynamic driving forces playing only a supportive role. In all signaling reactions, receptors use their catalytic activities to function as molecular am- plifiers. Directly or indirectly, a receptor gener- ates a chemical signal that is huge, both energetically and with respect to the number of molecules recruited by a single receptor. Molecular amplification is a hallmark of recep- tors and many other steps in cellular signaling pathways. Ligand binding changes receptor conformation A central mechanistic question in receptor func- tion is how the binding of a signaling molecule to the ligand-binding domain increases the ac- tivity of the effector domain. The key to this question is that receptors can exist in multiple molecular conformations, some active for sig- naling and others inactive. Ligands shift the conformational equilibrium among these con- formations. The structural changes that occur during the receptor’s inactive-active isomeriza- tion and how ligand binding drives these changes are exciting areas of biophysical re- search. However, the basic concept can be de- scribed simply in terms of coupling the conformational isomerizations of the ligand- binding and effector domains. Key concepts • Receptors can exist in active or inactive conformations. • Ligand binding drives the receptor toward the active conformation. 14.5 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 593
594 CHAPTER 14 Principles of cell signaling How do ligands activate (or not activate) a receptor? Most of the basic regulatory activities of receptors can be described by a simple scheme that considers the receptors as having two in- terconvertible conformations, inactive (R) and active (R*). R and R* are in equilibrium, which is described by the equilibrium constant J. Because unliganded receptors are usually minimally active, J<<1 and an unliganded recep- tor spends most of its time in the R state. When a signaling molecule (L) binds, it drives the re- ceptor toward the active conformation, R*, in which the effector domain is functional. The lig- and-bound receptor thus spends most of its time in the active R* state. The mechanism whereby a ligand can ac- tivate receptor is a simple consequence of its relative affinities for the receptor’s active and inactive conformations. A ligand can bind to the receptor in either of its conformations, de- scribed here by association constants K for the R state and K* for the R* state. Any ligand that binds with higher affinity for the R* conforma- tion than for R will be an activator. If K* is greater than K, the ligand is an agonist. According to the Second Law of Thermodynamics, a system of R + L J R*+ L R L J* K*K R* L R J R* coupled equilibria displays path independence: the net free energy difference between two states is independent of which intermediary re- actions take place. For the receptor, any path from R to R*L therefore has the same free en- ergy change, and the products of the equlib- rium constants along each path are equal. For the example above, path independence means that: J•K* = K•J* Therefore, J* / J = K* / K. Thus, if binding to the R* configuration is preferred (i.e., K*/K>>1), then ligand binding will shift the conformation to the R* state to an equivalent extent (i.e., J*/J>>1). The relative activation by a saturating concentration of lig- and, J*/J, will exactly equal the ligand’s relative selectivity for the active receptor conformation, K*/K. This argument is generally valid for the reg- ulation of a protein’s activity by any regulatory ligand. This model explains many properties of re- ceptors and their ligands both simply and quan- titatively. • First, J must be greater than zero for the equilibrium to exist. Thus, even unli- ganded receptor has some activity. Overexpressed receptors frequently dis- play their intrinsic low activity. • Because physiological receptors are nearly inactive in the absence of ligand, J must be much less than 1 and is prob- ably less than 0.01; most receptors are less than 1% active without agonist. • Ligands can vary in their selectivities between R and R*. Their abilities to ac- tivate will also vary. Some ligands, re- ferred to as agonists, can drive formation of appreciable R*. Others, known as par- tial agonists, will promote submaxi- mal activation. Chemical manipulation of a ligand’s structure will often alter its activity as an agonist. These relation- ships are depicted graphically in FIGURE 14.3. • A ligand that binds equally well to both the R and R* states will not cause acti- vation. However, such a ligand may still occupy the binding site and thereby competitively inhibit binding of an ac- tivating ligand. Such competitive in- hibitors, referred to as antagonists, are frequently used as drugs to block un- wanted activation of a receptor in var- ious disease states. • A ligand that binds preferentially to R 0 1.0 0.8 0.6 0.4 0.2 0 0.012 0.010 0.008 0.006 0.004 0.002 Log [L] Partial agonist Log [L] High affinity agonist Lower affinity agonist Fractional activity of receptor Fractional activity of receptor Inverse agonist Receptor ligands can vary in their activities and potencies FIGURE 14.3 The simple two-state model shown here can describe a wide va- riety of behaviors displayed by receptors and their various regulatory ligands. The left panel shows fractional activity of a receptor exposed to two agonists with different affinities and one partial agonist. The right panel shows the ef- fect of an inverse agonist. If the low fractional activity of unliganded receptor is detected as significant biological activity, then its inhibition by the inverse agonist would be easily detectable. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 594
14.6 Signals are sorted and integrated in signaling pathways and networks 595 relative to R* will further shift the con- formational equilibrium to the inactive state and cause net inhibition. Such lig- ands are called inverse agonists. Because J is already low, effects of in- verse agonists may only be noticeable if a receptor is overexpressed or if the receptor is mutated to increase its in- trinsic activity (i.e., the mutation in- creases J). • The extent to which an agonist stimu- lates a receptor is unrelated to its affin- ity. Both agonists and antagonists may bind with either high or low affinity. Affinity does determine the receptor’s sensitivity—that is, how low a concen- tration of ligand can the receptor detect. Affinities of receptors for natural regu- latory ligands vary enormously, with physiologic Kd values ranging from <10-12 M for some hormones to about 10-3 M for some bacterial chemoattrac- tants. Another aspect of sensitivity is how abruptly or gradually the receptor is activated as the concentration of ag- onist increases. The above model pre- dicts that a receptor is activated significantly at agonist concentrations between 0.1 and 10 times its Kd. A va- riety of cellular mechanisms can con- vert such a conventional response range of about 100-fold to either a more grad- ual response or a very steep, switchlike response. • This model only describes equilibria. It makes no predictions about the rates of ligand binding or release, or of the con- formational isomerization that leads to activation. This model shows how three important as- pects of receptor action are independently de- termined. As mentioned above, affinity for ligand, which determines the concentration range over which the ligand functions, is inde- pendent of the ligand’s net effectiveness at driv- ing receptor activation. The rate of response is also largely independent of these other two properties. Each aspect of receptor function can thus be independently regulated in response to other incoming signals or by the metabolic or developmental state of the cell. Such control of signal input is central to whole-cell coordina- tion of signal transduction. Examples and mech- anisms will recur throughout this chapter. Signals are sorted and integrated in signaling pathways and networks Receptors rarely act directly on the intracellu- lar processes that they ultimately regulate. Rather, receptors typically initiate a sequence of regulatory events that involve intermediary proteins and small molecules. The use of mul- tistep signaling pathways allows cells to amplify signals, adjust signaling kinetics, insert control points, integrate multiple signals, and route sig- nals to distinct effectors. Branched pathways give cells the ability to integrate multiple incoming signals and to di- rect information to the correct control points. As FIGURE 14.4 illustrates, branching can be ei- ther convergent, with multiple signals regulat- ing common end points, or divergent, with a single pathway branching to control more than one process. In multicellular organisms, diver- gent branching allows a single hormone recep- tor to initiate distinct cell-appropriate patterns of responses in different cells and tissues. Divergent signaling also allows a receptor to regulate qualitatively different cellular responses with quantitatively distinct intensities, each de- pendent on signal amplification in the interme- diary pathway. Convergent branching—when several re- ceptors activate the same pathway to elicit the same regulatory responses—is also common. Convergent branching allows multiple incom- ing signals, both stimulatory and inhibitory, to be integrated and coordinately regulated at a common site downstream of the receptors. Receptors for several different hormones fre- quently initiate similar or overlapping patterns of signaling in a single target cell. Overlapping converging and diverging sig- naling pathways create signaling networks within cells that coordinate responses to multiple in- puts (Figure 14.4). Typically, such pathways are complex in the number and diversity of their components and in the topology of their circuit Key concepts • Signaling pathways usually have multiple steps and can diverge and/or converge. • Divergence allows multiple responses to a single signal. • Convergence allows signal integration and coordination. 14.6 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 595
596 CHAPTER 14 Principles of cell signaling maps. Signaling networks are also spatially com- plex. They may include components in various subcellular locations, with initial receptors and associated proteins in the plasma membrane, but with downstream proteins in the cytoplasm or in- tracellular organelles. Such complexity is neces- sary to allow the cells to integrate and sort incoming signals and to regulate multiple intra- cellular functions simultaneously. The complexity and adaptability of signal- ing networks, like the one shown in the lower half of Figure 14.4, make their dynamics at the whole-cell level difficult or impossible to grasp intuitively. Signaling networks resemble large analog computers, and investigators are increas- ingly depending on computational tools to un- derstand cellular information flow and its regulation. First, many signaling interactions that include only two or three proteins exert functions analogous to traditional computa- tional logic circuits (see the next section). The theory and experience with such circuits in elec- tronics facilitate understanding biological sig- naling functions as well. The enormous complexity of cellular signal- ing networks can be simplified by considering them to be composed of interacting signaling modules, i.e., groups of proteins that process sig- nals in well-understood ways. A cellular signal- ing module is analogous to an integrated circuit in an electronic instrument that performs a known function, but whose exact components could be changed for similar use in another de- vice. The concept of modular construction facil- itates both qualitative and quantitative understanding of signaling networks. We will re- fer to many standard signaling modules later in the chapter. Examples include monomeric and heterotrimeric G protein modules, MAPK cas- cades, tyrosine (Tyr) kinase receptors and their binding proteins, and Ca2+ release/uptake mod- ules. In each case, despite the numerous phylo- genetic, developmental, and physiologic variations, understanding the basic function of that class of module conveys understanding of all its incarnations. Last, the evolutionary impor- tance of modules is significant; once the architec- ture of a module is established it can be reused. For larger-scale networks, multiplexed, high-throughput measurements on living cells have been combined with powerful kinetic mod- eling strategies to allow an increasingly accurate quantitative depiction of information flow within signaling modules or entire networks. Such models, with sound and experimentally based parameter sets, can describe signaling processes in systems too complex for intuitive or ad hoc analysis. They are also vital as tests of understanding because they can predict exper- imental results in ways that can be used to test the validity of the model. Well-grounded mod- els can then be used (cautiously) to suggest the mechanisms of systems for which data sets re- main unattainable. At even greater levels of complexity, the theories and tools of computer science are increasingly giving useful systems- level analyses of signal flow in cells. Using com- putational tools to analyze large arrays of quantitative data allows us to understand cel- lular information flow and its regulation. Linear, parallel Convergent Divergent Multiply branched RECEPTORS TRANSDUCERS EFFECTORS Convergent and divergent signaling pathways FIGURE 14.4 Signaling pathways use convergent and divergent branching to co- ordinate information flow. The diagrams at top show how even a simple, three- level signaling network can sort information. Convergence or divergence can take place at multiple points along a signaling pathway. As an example of com- plexity, the lower portion of the figure shows a small segment (~10%) of the G protein-mediated signaling network in a mouse macrophage cell line. It omits several interpathway regulatory mechanisms and completely ignores inputs from non-G protein-coupled receptors. Pathway map courtesy of Lily Jiang, University of Texas Southwestern Medical Center. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 596
14.7 Cellular signaling pathways can be thought of as biochemical logic circuits 597 Developing quantitative models of signaling networks is a frontier in signaling biology. These models both help describe network function and pinpoint experiments to clarify mechanism. Cellular signaling pathways can be thought of as biochemical logic circuits As introduced in the preceding section, processes thatsignalingpathwaysusetointegrateanddirect information to cellular targets are strikingly anal- ogoustothemathematicallogicfunctionsthatare used to design the individual circuits of electronic computers. Indeed, there are biological equiva- lents of essentially all of the functional compo- nents that computer scientists and engineers considerinthedesignofcomputersandelectronic control devices. To understand signaling path- ways, it is, therefore, useful to consider groups of reactionswithinapathwayasconstitutinglogiccir- cuits of the sort used in electronic computing, as illustrated in FIGURE 14.5. The simplest example is whentwostimulatorypathwaysconverge.Ifsuf- ficient input from either is adequate to elicit the response, the convergence would constitute an “OR” function. If neither input is sufficient by it- self but the combination of the two elicits the re- sponse, then the converging pathways would create “AND” functions. AND circuits are also re- ferredtoascoincidencedetectors—aresponse is elicited only when two stimulating pathways are activated simultaneously. AND functions can result from the combi- nation of two similar but quantitatively inade- quate inputs. Alternatively, two mechanistically different inputs might both be required to elicit a response. An example of the latter would be a target protein that is allosterically activated only when phosphorylated, or that is activated by phosphorylation but is only functional when recruited to a specific subcellular location. The opposite of an AND circuit is a NOT function, where one pathway blocks the stim- Key concepts • Signaling networks are composed of groups of biochemical reactions that function as mathematical logic functions to integrate information. • Combinations of such logic functions combine as signaling networks to process information at more complex levels. 14.7 ulatory effect of another. Simple logic gates are observed at many locations in cellular signaling pathways. We can also think about convergent signal- ing in quantitative rather than Boolean terms by considering the additivity of inputs to a dis- tinct process (see Figure 14.5, right). The OR function referred to above can be considered to be the additive positive inputs of two pathways. Such additivity could represent the ability of several receptors to stimulate a pool of a partic- ular G protein or the ability of two protein ki- nases to phosphorylate a single substrate. Additivity may be positive, as in the examples above, or negative, such as when two inhibitory inputs combine. Inhibition and stimulation may also combine additively to yield an algebraically balanced output. Alternatively, multiple inputs can combine with either more or less than an additive effect. The NOT function, discussed above, is analogous to describing a blockade of stimulation. The AND function describes syn- ergism, where one input potentiates another but alone has little effect. Even simple signaling networks can display complex patterns of information processing. One Additive Logical (Boolean) Quantitative (Analog) Response A + B Response A OR B A NOT B A AND B B A Response Response B A A + fixed [B] A + B A Response Response A + B B A Less than additive More than additive A + B Response B A A + B B A Response log (agonist concentration) log (agonist concentration) log (agonist concentration) B Simple logic circuits FIGURE 14.5 Signaling networks use simple logic functions to process information. Boolean OR, AND, and NOT functions (left) correspond to the quantitative interactions between converging signals that are shown on the right. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 597
598 CHAPTER 14 Principles of cell signaling good example is the creation of “memory”: mak- ing the effect of a transient signal more or less permanent. Signaling pathways have multiple ways of setting memories, and of forgetting. One mechanism, common in protein kinase path- ways, is the positive feedback loop, illustrated in the top panel of FIGURE 14.6. In a positive feed- back loop, the input stimulates a transducer (T), which in turn stimulates the effector protein (E) to create the output. If the effector can also ac- tivate the transducer, sufficient initial signal can be fed back to the transducer that it can main- tain the effector's full signal output even when input is removed. Such systems typically display a threshold behavior, as shown on the right. A positive feed-forward loop can generate memory of another type (Figure 14.6, middle panel), indicating the duration of input. In such circuits, the effector requires simultaneous in- put from both the receptor and from the inter- mediary transducer. If the pathway from receptor through transducer is relatively slow, or if it requires the accumulation of a substan- tial amount of transducer, only a prolonged in- put will trigger a response, as shown in the time-base output diagram at the right. A third way to establish memory is to allow one input to control the reversibility of a sec- ond regulatory event (Figure 14.6, bottom panel). WASP, a protein that initiates the polymerization of actin to drive cellular motion and shape change, is activated both by phosphorylation and by the binding of Cdc42, a small GTP-bind- ing protein (G). However, the phosphorylation site on WASP is only exposed when WASP is bound to Cdc42. Phosphorylation thus requires both activated Cdc42 and activated protein ki- nase. If Cdc42 dissociates, the phosphorylated state of WASP persists until another signaling molecule, whose identity remains uncertain, binds again to expose the site to a protein phos- phatase. As shown in the time-base graph, ex- posure to Cdc42 will activate, but exposure to kinase alone will not. If Cdc42 is present, then the kinase can activate WASP. Phospho-WASP is relatively insensitive to protein phosphatase (P) alone, but can be dephosphorylated if Cdc42 or another G protein binds to expose the site to phosphatase. Scaffolds increase signaling efficiency and enhance spatial organization of signaling Key concepts • Scaffolds organize groups of signaling proteins and may create pathway specificity by sequestering components that have multiple partners. • Scaffolds increase the local concentration of signaling proteins. • Scaffolds localize signaling pathways to sites of action. 14.8 Positive feedback loop : irreversible ON switch Positive feed-forward loop : responds to prolonged input Conformational lock - Dual control switch Input Input strength Output Output T Input OutputT Output Time Time Output + + input Kinase Phosphatase OH G OH G P G P G P G K PG K G P E E OH E E E E E E Signal processing circuits FIGURE 14.6 Relatively complex signal processing can be executed by simple multi-protein modules. The figure depicts three types of signaling modules (left) and their behavior in response to agonist (right). (top) In a positive feed-back module, a transducer protein (T) stimulates an effector (E) to pro- duce a cellular output, but the effector also stimulates the activity of the trans- ducer. The result can be an all-or-none switch, where input up to a threshold has little effect, but then becomes committed when feedback from the effec- tor is sufficient to maintain transducer activity even in the absence of contin- ued input from the receptor. (center) In a positive feed-forward module, the effector requires input both from the transducer and from upstream in the path- way. When stimulation is brief (short horizontal bar under trace at right), sig- nificant amounts of active transducer do not accumulate and output is minimal. When stimulation is prolonged (longer bar), signal output is substantial. (bot- tom) In some dual-control switching modules, the binding of one regulator (G) can both activate the effector and expose another regulatory site, shown here as a Ser substrate site (-OH) for a protein kinase. The effector can only be phos- phorylated or dephosphorylated when G is bound. Therefore, as shown at the right, addition of G alone will activate but activation of the kinase (K) alone will not. If kinase is active while G is bound, phosphorylation is resistant to phosphatase activity unless G is again present to reexpose the phosphoserine residue (shown on the graph at the right as a bold P). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 598
14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling 599 The proteins in a signaling pathway are fre- quently colocalized within cells such that their mutual interactions are favored and their in- teractions with other proteins are minimized. Many signaling pathways are organized on scaf- folds. Scaffolds bind several components of a signaling pathway in multiprotein complexes to enhance signaling efficiency. Scaffolds pro- mote interactions of proteins that have a low affinity for each other, accelerate activation (and often inactivation) of the associated compo- nents, and localize the signaling proteins to ap- propriate sites of action. Colocalization may be tonic or regulated, and stimulus-dependent scaf- folding often determines signaling outputs. The binding sites on a scaffolding protein are often localized in distinct modular protein- binding domains, giving the impression that the protein is designed simply to hold the compo- nents of the pathway together. Many scaffold- ing proteins do lack intrinsic enzymatic activity, but some signaling enzymes also act as scaffolds. Binding to a scaffold facilitates signaling by increasing the local concentrations of the com- ponents, so that diffusion or transport of mol- ecules to their sites of action is not necessary. In the photoreceptor cells of Drosophila, scaffold- ing of signaling components is critical for rapid signal transmission. These cells contain the InaD scaffolding protein, which has five modular binding domains, known as PDZ domains. Each of its PDZ domains binds to a C-terminal motif of a target protein, thereby facilitating interac- tions among the associated proteins. FIGURE 14.7 shows a model for how InaD organizes the sig- naling proteins. The mutational loss of InaD produces a nearly blind fly, and deletion of a single PDZ domain can yield a fly with a dis- tinct visual defect characteristic of the protein that binds to the missing domain. A second example is Ste5p, a scaffold for the pheromone-induced mating response pathway in S. cerevisiae. FIGURE 14.8 illustrates how Ste5p binds and organizes components of a mitogen- PKC CYTOSOL TRP Rhodopsin CaMCaM PDZ PDZ - PDZ PKC PDZ - PDZ INAD PDZZ INAD PDZPDZ PDZ PDZ PDZ The INAD signaling complex FIGURE 14.7 The scaffold InaD organizes proteins that transmit visual signals in the fly photoreceptor cell. InaD is localized to the photorecep- tor membrane and coordinates light sensing and visual transduction. In invertebrate eyes, the visual signaling pathway goes from rhodopsin through Gq to a phospholipase C-␤, and Ca2+ release triggered by PLC ac- tion initiates depolarization. This system is specialized for speed, and re- quires that the relevant proteins are nearby. InaD contains five PDZ domains, each of which binds to the C terminus of a signal transducing protein. The TRP channel, which mediates Ca2+ entry, PLC-␤, and a pro- tein kinase C isoform that is involved in rapid desensitization all bind con- stitutively to InaD. Rhodopsin and a myosin (NinaC) also bind, and Gq binds indirectly. Scaffold determines specificity of Ste11p signaling Scaffold organizes MAPK cascade Ste11p Ste11p Ste20p Ste7p Ste7p Fus3p Fus3p Ste20p Ste20pSte20p Ste20pSte20p Ste7p Fus3p Ste11p Pheromone Cdc42pCdc42p Cdc42pCdc42p Mating response G protein Ste5p Ste5p Pbs2p Pheromone High osmolarity Osmo- adaptation Mating response Ste11p Hog1p Scaffolds concentrate and insulate signaling proteins GPCR FIGURE 14.8 The scaffold Ste5p organizes the components of the MAPK cascade that mediates the pheromone-induced mating response in Saccharomyces cerevisiae. In the top left panel, Ste5p brings the compo- nents of the MAPK cascade to the membrane in response to pheromone. In the top right panel, binding to the heterotrimeric G protein brings loaded Ste5p in proximity to the protein kinase Ste20p bound to the activated small GTP binding protein Cdc42p. Their colocalization facilitates the sequential activation of the cascade components, resulting in activation of the MAPK Fus3p and the mating response. The MAP3K Ste11p can regulate not only the MAPK Fus3p in the mating pathway, but also the MAPK Hog1p in the high osmolarity pathway, as shown in the bottom two panels. The scaffold to which Ste11p binds, either Ste5p or Pbs2 (both a scaffold and a MAP2K), determines which MAPK and downstream events are activated as the out- put. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 599
600 CHAPTER 14 Principles of cell signaling activated protein kinase (MAPK) cascade, in- cluding a MAP3K (Ste11p), a MAP2K (Ste7p) and a MAPK (Fus3p). (The MAPK cascade will be discussed in 14.32 MAPKs are central to many signaling pathways). The function of Ste5p is par- tially retained even if the positions of its bind- ing sites for the kinases are shuffled in the linear sequence of the protein, indicating that a major role is to bring the enzymes into proximity, rather than to precisely orient them. Ste5p also binds to the ␤␥ subunits of the heterotrimeric G pro- tein that mediates the actions of mating pheromones, linking the membrane signal to the intracellular transducers. Yeast that lack Ste5p cannot mate, demonstrating that Ste5p is required for this biological function (but not all functions) carried out by the pathway. In addition to facilitating signaling in their own pathways, scaffolds can enhance signaling specificity by limiting interactions with other signaling proteins. Scaffolds thus insulate com- ponents of a signaling pathway both from acti- vation by inappropriate signals and from producing incorrect outputs. For example, the mating and osmosensing pathways in yeast share several components, including the MAP3K Ste11p, but each pathway maintains specificity because it employs different scaffolds that restrict signal transmission. In contrast, the presence of excess scaffold can inhibit signaling because the individual sig- naling components will more frequently bind to distinct scaffold proteins rather than forming a functional complex. Such dilution among scaf- folds causes separation rather than concentra- tion of the components, preventing their productive interaction. Independent, modular domains specify protein- protein interactions Modular protein interaction domains or motifs occur in many signaling proteins and confer the ability to bind structural motifs in other mole- cules, including proteins, lipids, and nucleic Key concepts • Protein interactions may be mediated by small, conserved domains. • Modular interaction domains are essential for signal transmission. • Adaptors consist exclusively of binding domains or motifs. 14.9 acids. Some of these domains are listed in FIGURE 14.9. In contrast to scaffolds, which bind spe- cific proteins with considerable selectivity, mod- ular interaction domains generally recognize not a single molecule but a group of targets that share related structural features. Modular interaction domains important for signal transduction were first discovered in the protein tyrosine kinase proto-oncogene Src, which contains a protein tyrosine kinase do- main and two domains named Src homology (SH) 2 and 3 domains. The modular SH2 and SH3 domains were originally identified by com- parison of Src to two other tyrosine kinases, Fps and Abl. One or both of these domains appear in numerous proteins and both are critically in- volved in protein-protein interactions. SH3 domains, which consist of approxi- mately 50 residues, bind to specific short pro- line-rich sequences. Many cytoskeletal proteins and proteins found in focal adhesion complexes contain SH3 domains and proline rich se- quences, suggesting that this targeting motif may send proteins with these domains to these sites of action within cells. In contrast to phos- photyrosine-SH2 binding, the proline-rich bind- ing sites for SH3 domains are present in resting and activated cells. However, SH3-proline inter- actions may be negatively regulated by phospho- rylation within the proline-rich motif. SH2 domains, which consist of approxi- mately 100 residues, bind to Tyr phosphory- lated proteins, such as cytoplasmic tyrosine kinases and receptor tyrosine kinases. Thus, Tyr phosphorylation regulates the appearance of SH2 binding sites and, thereby, regulates a set of protein-protein interactions in a stimulus- dependent manner. A clever strategy was used to identify the binding specificity of SH2 domains. An isolated recombinant SH2 domain was incubated with cell lysates and then recovered from the lysates using a purification tag. The proteins associated with the SH2 domain were some of the same proteins that were recognized by antiphospho- tyrosine antibodies. By this and other methods, it was discovered that SH2 domains recognize sequences surrounding Tyr phosphorylation sites and require phosphorylation of the in- cluded Tyr for high affinity binding. Information on specific amino acid se- quences that recognize and bind to modular binding domains is being accumulated as these individual interactions are identified. In addi- tion, screening programs using cDNA and/or peptide libraries to assess binding capabilities 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 600
14.9 Independent, modular domains specify protein-protein interactions 601 Characteristics of some common modular protein domains 14-3-3 Binds protein phosphoserine or phosphothreonine Protein sequestration Domain Characteristics Cellular involvement WW Binds proline-rich sequences Alternative to SH3; vesicular trafficking TPR Degenerate sequence of ~34 amino acids with residues WL/GYAFAP; forms a scaffold Wide variety of processes SH3 Binds to PXXP motifs Various processes SH2 Binds to protein phosphotyrosine (pY) Tyrosine protein kinase signaling SAM Homo- and hetero- oligomerization Wide variety of processes RING Binds zinc and may be found in E3 ubiquitin ligases Ubiquitination, transcription PH Binds to specific phosphoinositi- des, esp. PI-4,5-P2, PI-3,4-P2 or PI-3,4,5-P3. Recruitment to mem- branes and motility PDZ Binds to the C-terminal 4-5 residues of proteins that have a hydrophobic residue at the terminus; may bind to PIP2 Scaffolding diverse protein complexes often at the membrane LIM Zinc-binding cysteine-rich motif that forms two tandemly repeated zinc fingers Wide variety of processes HECT Binds E2 ubiquitin-conjugating enzymes to transfer ubiquitin to the substrate or to ubiquitin chains Ubiquitination FYVE Binds to PI(3)P Membrane trafficking, TGF-␤ signaling FHA Binds protein phosphothreonine or phosphoserine Various; DNA damage F-Box Binds Skp1 in a ubiquitin-ligase complex Ubiquitination EF hand Binds calcium Calcium-dependent processes C2 Binds phospholipids Signal transduction, vesicular trafficking C1 Binds phorbol esters or diacyl- glycerol Recruitment to mem- branes Dimerization Caspase activation Bromo CARD Binds acetylated lysine residues Chromatin-associated proteins FIGURE 14.9 The table describes a subset of known modular protein in- teraction domains found in many proteins. Interactions mediated by these domains are essential to controlling cell function. Few if any of these do- mains exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/). yield such motifs. Consensus target sequences for individual domains have been identified based on the sequence specificity of their bind- ing to arrayed sequences. These consensus se- quences can then be used to predict whether the domain will bind a site in a candidate pro- tein. Adaptor proteins, which lack enzymatic activity, link signaling molecules and target them in a manner that is responsive to extra- cellular signals. Adaptor proteins are generally made up of two or more modular interaction domains or the complementary recognition motifs. Unlike scaffolds, adaptors are usually 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 601
602 CHAPTER 14 Principles of cell signaling multifunctional because their modular interac- tion domains and motifs are not as highly spe- cific. Adaptors bind to two or more other signaling proteins via their protein-protein in- teraction domains to colocalize them or to fa- cilitate additional interactions. Grb2 is a prototypical adaptor protein that was identified as a protein that bound to the C- terminal region of the EGF receptor. Grb2 has one SH2 and two SH3 domains. It binds consti- tutively to specific proline-rich segments of pro- teins through its SH3 domain, although this binding can be negatively regulated. One target of Grb2 is SOS, a guanine nucleotide exchange factor that activates the small GTP-binding pro- tein Ras in response to EGF signaling. Through its SH2 domain, Grb2 binds Tyr-phosphorylated proteins, including the receptors themselves in a stimulus-dependent manner. Thus, Tyr phos- phorylation of these receptors in response to ligand will enable the binding of Grb2 to the re- ceptors, which, in turn, will recruit SOS to the membrane-localized receptor. Once at the mem- brane, SOS can activate its target, Ras. Cellular signaling is remarkably adaptive Auniversalpropertyofcellularsignalingpathways is adaptation to the incoming signal. Cells contin- uously adjust their sensitivity to signals to main- taintheirabilitytodetectchangesininput.Typically, when a cell is exposed to a new input, it initiates a process of desensitization that dampens the cel- lularresponsetoanewplateaulowerthantheini- tial peak response, as illustrated in FIGURE 14.10. When the stimulus is removed, the desensitized state can persist, with sensitivity slowly returning to normal. Similarly, the removal of a tonic stim- ulus can hypersensitize signaling systems. Key concepts • Sensitivity of signaling pathways is regulated to allow responses to change over a wide range of signal strengths. • Feedback mechanisms execute this function in all signaling pathways. • Most pathways contain multiple adaptive feedback loops to cope with signals of various strengths and durations. 14.10 Initial response Heterologous desensitization Homologous desensitization Time Time R1 R2 R2 X1 X2 Y Z Response R esponse a b K a R1 R2 Z X1 X2 Agonist Agonist Agonist Desensitization Agonist a for R1 Reapply a or b Agonist a for R1 Reapply a or b Time Response R1R1 R2R1 or a Y Patterns of adaptation in signaling networksFIGURE 14.10 Top: Upon exposure to a stimulus, signaling pathways adjust their sensitivities to adapt to the new level of input. Thus, the response de- cays after initial stimulation. A sec- ond similar stimulus will elicit a smaller response unless adequate time is al- lowed for recovery. Bottom: Some adap- tation mechanisms feed back only on the receptor that is stimulated and do not alter parallel pathways. Such mech- anisms are referred to as homologous. At left, agonist a for receptor R1 can initiate either of two feedback events that desensitize R1 alone. In other cases, a stimulus will also cause par- allel or related systems to desensitize. At the right, agonist a initiates desen- sitization of both R1 and R2. The re- sponse to agonist b, which binds to R2, is also desensitized. Such heterol- ogous desensitization is common. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 602
14.10 Cellular signaling is remarkably adaptive 603 Adaptation in signaling is one of the best ex- amples of biological homeostasis. The adaptabil- ity of cellular signaling can be quite impressive. Cells commonly regulate their sensitivity to phys- iological stimuli over more than a 100-fold range, and the mammalian visual response can adapt to incoming light over a 107-fold range. This re- markable ability allows a photoreceptor cell to detect a single photon, and allows a person to read in both very dim light and intense sunlight. Adaptability is observed in bacteria, plants, fungi, and animals. Many of its properties are conserved throughout biology, although the most complex adaptive mechanisms are found in animals. The general mechanism for adaptation is the nega- tive feedback loop, which biochemically samples the signal and controls the adaptive process. Adaptationvarieswithboththeintensityand the duration of the incoming signal. Stronger or more persistent inputs tend to drive greater adap- tive change and, often, adaptation that persists for a longer time. Cells can modulate adaptation in this way because adaptation is exerted by a successionofindependentmechanisms,eachwith its own sensitivity and kinetic parameters. G protein pathways offer excellent examples of adaptation. FIGURE 14.11 shows that the earli- est step in adaptation is receptor phosphoryla- tion, which is catalyzed by G protein-coupled receptor kinases (GRKs) that selectively recog- nize the receptor’s ligand-activated conforma- tion.Phosphorylationinhibitsthereceptor’sability to stimulate G protein activation and also pro- motes binding of arrestin, a protein that further inhibits G protein activation. Moreover, arrestin binding primes receptors for endocytosis, which removes them from the cell surface. Endocytosis can also be the first step in receptor proteolysis. Along with these direct effects, many receptor genes display feedback inhibition of transcrip- tion, such that signaling by a receptor decreases its own expression. Stimulation thus causes multiple adaptive processes that range from immediate (phospho- rylation, arrestin binding) through delayed (tran- scriptionalregulation),andincludebothreversible and irreversible events. This array of adaptive events has been demonstrated for many G pro- tein-coupled receptors, and many cells may use all of them to control output from one receptor. The speed, extent, and reversibility of adaptation are selected by a cell’s developmental program. Cells can change their patterns of adaptation both qualitatively and quantitatively by altering the points in a pathway where feedback is initi- ated and exerted. In a linear pathway, changing DNA GRK Relative response Agonist added Endosomal receptor degradation Receptor transcription inhibited Receptor phosphorylation Arrestin binding Receptor endocytosis G protein Early endosome Lysosome Time (seconds) 0 1 10 100 1000 Agonist binds G protein active EFFECTORS Arrestin GPCR G P C R gene 1 2 3 5 4 5 Receptor transcription inhibited C Y T O P L A S M N U C L E U S Agonist Receptor degradation 4 Receptor endocytosis 3 Receptor recycling 1 Receptor phosphorylation Arrestin binding 2 Multiple adaptation processes occur after a stimulus FIGURE 14.11 Multiple adaptation processes are invoked during a stimulus, and multiple nested mechanisms for adaptation are the rule. They are usually invoked sequentially according to the duration and intensity of the stimulus. For GPCRs, at least five desensitizing mechanisms are known, with others act- ing on the G protein and effectors. these points will alter the kinetics or extent of adaptation(Figure14.10).Inbranchedpathways, changing these points can determine whether adaptation is unique to one input or is exerted formanysimilarinputs.Ifreceptoractivationtrig- gers its desensitization directly, or if an event downstream on an unbranched pathway triggers desensitization,thenonlysignalsthatinitiatewith that receptor will be altered. Receptor-selective adaptation is referred to as homologous adap- tation (Figure 14.10). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 603
604 CHAPTER 14 Principles of cell signaling Alternatively, feedback control can initiate downstream from multiple receptors in a con- vergent pathway and thus regulate both the initiating receptor and the others. Such het- erologous adaptation regulates all the possi- ble inputs to a given control point. A common example is the phosphorylation of G protein- coupled receptors by either protein kinase A or protein kinase C, which are activated by down- stream signals cAMP or Ca2+ plus the lipid dia- cylglycerol, respectively. Like GRK, these kinases both attenuate receptor activity and promote arrestin binding. Cells also alter their responses to incoming signals for homeostatic reasons. These consid- erations include phase of the cell cycle, meta- bolic status, or other aspects of cellular activity. Again, all these adaptive processes may be dis- played to a greater or lesser extent in different cells, different pathways within a cell or differ- ent situations during the cell’s lifetime. Signaling proteins are frequently expressed as multiple species Key concepts • Distinct species (isoforms) of similar signaling proteins expand the regulatory mechanisms possible in signaling pathways. • Isoforms may differ in function, susceptibility to regulation or expression. • Cells may express one or several isoforms to fulfill their signaling needs. 14.11 Cells increase the richness, adaptability, and regulation of their signaling pathways by ex- pressing multiple species of individual signal- ing proteins that display distinct biochemical properties. These species may be encoded by multiple genes or by multiple mRNAs derived from a single gene by alternative splicing or mRNA editing. The numerical complexity im- plicit in these choices is impressive. Consider the neurotransmitter serotonin: In mammals, there are thirteen serotonin receptors, each of which stimulates a distinct spectrum of G pro- teins of the Gi, Gs, and Gq families. (A four- teenth serotonin receptor is an ion channel.) FIGURE 14.12 shows the relationship of serotonin receptors to these G protein families. There is also tremendous diversity among the G proteins and adenylyl cyclases. There are three genes for Gαi and one each for the closely related Gαz and Gαo. Furthermore, the Gαo mRNA is multiply spliced. There are four Gq members. In addition, there are five genes for Gβ and twelve for Gγ, and most of the possible Gβγ dimers are expressed naturally. There are ten genes for adenylyl cyclases, which are direct targets of Gs and either direct or indirect targets of the other G proteins. While all nine mem- brane-bound adenylyl cyclase isoforms are stim- ulated by Gαs, they display diverse stimulatory and inhibitory responses to Gβγ, Gαi, Ca2+, calmodulin, and several protein kinases, as il- lustrated in FIGURE 14.13. Thus, stimulation by serotonin can lead to diverse responses depend- ing upon the various forms of the proteins that are engaged at a particular time and location. FIGURE 14.12 Receptors for serotonin have evolved in mammals as a family of 13 genes that regulate three of the four major classes of G pro- teins. While all respond to the natural ligand serotonin, the binding sites have evolved suf- ficient differences that drugs have been devel- oped that specifically target one or more isoforms. The type 3 serotonin receptors, not shown here, are ligand-gated ion channels and are not obviously related to the others. 1B Gi Gs Gs Gq 1D 1E 1F 1A 7 5A 5B 4 2A 2C 2B 6 120 100 80 60 40 20 0 Isoforms Nucleotide substitution distance G protein Evolutionary relationship of serotonin receptor isoforms 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 604
14.12 Activating and deactivating reactions are separate and independently controlled 605 Sometimes isoforms of a signaling protein are subject to quite different kinds of inputs. For example, all of the members of the phospho- lipase C family (PLC) hydrolyze phosphatidyli- nositol-4,5-bisphosphate to form two second messengers, diacylglycerol and inositol-1,4,5 trisphosphate (see 14.16 Lipids and lipid-derived compounds are signaling molecules). The distinct isoforms may be regulated by diverse combina- tions of Gαq, Gβγ, phosphorylation, monomeric G proteins, or Ca2+. Because a cell has multiple options when expressing a form of a signaling protein, it can use expression of particular isoforms to alter how it performs otherwise identical signaling functions. Different cells express one or more isoforms to allow appropriate responses, and ex- pression can vary according to other inputs or the cell’s metabolic status. In addition, signaling pathways are remarkably resistant to mutational or other injuries because loss of a single species or isoform of a signaling protein can often be compensated for by increased expression or ac- tivity of another species. Similarly, engineered overexpression can result in the reduced expres- sion of endogenous proteins. The existence of multiple receptor species can, thus, substantially add to adaptability and the consequent resist- ance of signaling networks to damage. Activating and deactivating reactions are separate and independently controlled In signaling networks, individual proteins are frequently activated and deactivated by distinct reactions, a feature that facilitates separate reg- ulation. Common examples include using pro- tein kinases and phosphoprotein phosphatases Key concepts • Activating and deactivating reactions are usually executed by different regulatory proteins. • Separating activation and inactivation allows for fine-tuned regulation of amplitude and timing. 14.12 CaM CaMK Gαs Gαi Gβγ PKA PKC inhibit activate Regulators Ca2+ NO Different isoforms of adenylyl cyclase are regulated differently FIGURE 14.13 All of the mammalian membrane-bound adenylyl cyclases are structurally homologous and catalyze the same reaction, and all are stimulated by G␣s. Their responses to other inputs (protein kinases CaMK, PKA and PKC; Ca2+; calmodulin (CaM); NO•) are specific to each isoform, allowing a rich com- binatoric input to cellular cAMP signaling. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 605
606 CHAPTER 14 Principles of cell signaling to catalyze protein phosphorylation and de- phosphorylation; using adenylyl cyclase to cre- ate cAMP while using phosphodiesterases to hydrolyze it or anion transporters to pump it out of the cell; or using GTP/GDP exchange fac- tors (GEFs) to activate G proteins and GTPase- activating proteins (GAPs) to deactivate them. Depending on stoichiometry and detailed mech- anism, these strategies can convey either addi- tive or nonadditive inputs while maintaining fine control over the kinetics of activation and deactivation of a signaling pathway. The use of distinct reactions for activation and deactiva- tion is analogous to the use of distinct anabolic and catabolic enzymes in reversible metabolic pathways. Cellular signaling uses both allostery and covalent modification Cellular signaling uses almost every imagina- ble mechanism for regulating the activities of intracellular proteins, but most can be described as either allosteric or covalent. Individual sig- naling proteins typically respond to multiple al- losteric and covalent inputs. Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. Because a protein’s activity reflects its confor- mation, the binding of any molecule that alters conformation can change the target protein’s activity. Any molecule can have allosteric ef- fects: protons or Ca2+, small organic molecules, or other proteins. Allosteric regulation can be both inhibitory or stimulatory. Covalent modification of a protein’s chem- ical structure is also frequently used to regulate its activity. The change in the protein’s chemi- cal structure alters its conformation and, thus, its activity. Most regulatory covalent modifica- tion is reversible. The classic and most common regulatory covalent event is phosphorylation, in which a phosphoryl group is transferred from ATP to the protein, most often to the hydroxyl group of serine (Ser), threonine(Thr), or tyro- sine (Tyr). Enzymes that phosphorylate proteins Key concepts • Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. • Modification of a protein’s chemical structure is also frequently used to regulate its activity. 14.13 are known as protein kinases. Their actions are opposed by phosphoprotein phosphatases, which catalyze the hydrolysis of the phosphoryl group to yield free phosphate and restore the unmod- ified hydroxyl residue. Other forms of covalent modification are also common and will be ad- dressed throughout the chapter. Second messengers provide readily diffusible pathways for information transfer Signaling pathways make use of both proteins and small molecules according to their distinc- tive attributes. A small molecule used as an in- tracellular signal, or second messenger, has a number of advantages over a protein as a sig- naling intermediary. Small molecules can be synthesized and destroyed quickly. Because they can be made readily, they can act at high con- centrations so that their affinities for target pro- teins can be low. Low affinity permits rapid dissociation, such that their signals can be ter- minated promptly when free second messenger molecules are destroyed or sequestered. Because second messengers are small, they also can dif- fuse quickly within the cell, although many cells have developed mechanisms to spatially restrict such diffusion. Second messengers are, thus, superior to proteins in mediating fast responses, particularly at a distance. Second messengers are also useful when signals have to be addressed to large numbers of target proteins simultane- ously. These advantages often overcome their lack of catalytic activity and their inability to bind multiple molecules simultaneously. FIGURE 14.14 lists intracellular second mes- sengers developed through evolution. This num- ber is surprisingly low. Several are nucleotides synthesized from major metabolic nucleotide precursors. They include cAMP, cyclic GMP, ppGppp, and cyclic ADP-ribose. Other soluble second messengers include a sugar phosphate, inositol-1,4,5-trisphosphate(IP3),adivalentmetal ion Ca2+, and a free radical gas nitric oxide (NO•). Lipid second messengers include diacylglycerol and phosphatidylinositol-3,4,5-trisphosphate, Key concepts • Second messengers can propagate signals between proteins that are at a distance. • cAMP and Ca2+ are widely used second messengers. 14.14 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 606
14.14 Second messengers provide readily diffusible pathways for information transfer 607 phosphatidylinositol-4,5-diphosphate, sphingo- sine-1-phosphate and phosphatidic acid. The first signaling compound to be described as a second messenger was cAMP. The name arose because cAMP is synthesized in animal cells as a second, intracellular signal in response to numerous extracellular hormones, the first messengers in the pathway. cAMP is used by prokaryotes, fungi, and animals to convey in- formation to a variety of regulatory proteins. (Its occurrence in higher plants has still not been proved.) Adenylyl cyclases, the enzymes that syn- thesize cAMP from ATP, are regulated in vari- ous ways depending on the organism in which they occur. In animals, adenylyl cyclase is an integral protein of the plasma membrane whose multiple isoforms are stimulated by diverse agents (see Figure 14.13). In animal cells, adeny- lyl cyclase is generally stimulated by Gs, which was originally discovered as an adenylyl cyclase regulator. Some fungal adenylyl cyclases are also stimulated by G proteins. Bacterial cyclases are far more diverse in their regulation. cAMP is removed from cells in two ways. It may be extruded from cells by an ATP-driven anion pump but is more often hydrolyzed to 5′- AMP by members of the cyclic nucleotide phos- phodiesterase family, a large group of proteins that are themselves under multiple regulatory controls. The prototypical downstream regulator for cAMP in animals is the cAMP-dependent pro- tein kinase, but a bacterial cAMP-regulated tran- scription factor was discovered shortly thereafter, and other effectors are now known (Figure 14.14). The cAMP system remains the proto- typical eukaryotic signaling pathway in that its components exemplify almost all of the recog- nized varieties of signaling molecules and their interactions: hormone, receptor, G protein, adenylyl cyclase, protein kinase, phosphodi- esterase, and extrusion pump. The second messenger-stimulated protein kinase PKA is a tetramer composed of two cat- alytic (C) subunits and two regulatory (R) sub- units, as illustrated in FIGURE 14.15. The R subunit binds to the catalytic subunit in the substrate- binding region, maintaining C in an inhibited state. Each R subunit binds two molecules of cAMP, four cAMP molecules per PKA holoen- zyme. When these sites are filled, the R subunit dimer dissociates rapidly, leaving two free cat- alytic subunits with high activity. The difference in affinity of R for C in the presence and absence of cAMP is ~10,000-fold. The strongly cooper- ative binding of cAMP generates a very steep activation curve with an apparent threshold be- low which no significant activation of PKA oc- curs, as illustrated in Figure 14.15. PKA activity, thus, increases dramatically over a narrow range of cAMP concentrations. PKA is also regulated Protein kinase A Bacterial trans- cription factors Cation channel Cyclic nucleotide phospho- diesterase Rap GDP/GTP exchange factor (Epac) RNA polymerase ObgE trans- cription arrest detector IP3-gated Ca2+ channel Protein kinase C Trp cation channel Ion channel Transporters Protein kinase G Cation channel Cyclic nucleotide phospho- diesterase Ca2+ channel Various two component system proteins Guanylyl cyclase Numerous calmodulin Akt (protein kinase B) Other PH domains/proteins Adenylyl cyclase Rel1A SpoT Phospho- lipase C Phospho- lipase C PIP 5-kinase PI 3-kinase Guanylyl cyclase ADP-ribose cyclase Diguanylate cyclase NO. synthase ATP GTP PIP2 PIP2 PI-4-P GTP NAD GTP arginine Stored Ca2+ PIP2 Phospho- diesterase Organic anion transporter SpoT- catalyzed hydrolysis Phosphatase Diacylglycerol kinase Diacylglycerol lipase Phospho- lipase C Phosphatase Phospho- diesterase Hydrolysis Cyclic di-GMP phospho- diesterase Reduction Reuptake and extrusion pumps Phosphatase Magic spot 3':5'-cyclic AMP (cAMP) (ppGpp, ppGppp) Inositol-1,3,5- trisphosphate (IP3) Diacylglycerol (DAG) Phosphatidyl- inositol-4,5- bisphosphate (PIP2) 3':5'-Cyclic GMP (cGMP) Cyclic ADP-ribose Cyclic diguanosine- monophosphate Nitric oxide (NO.) Ca2+ Phosphatidyl- inositol-3,4,5- trisphosphate Second messenger Synthesis/ ReleaseTargets Pre- cursor Removal Release from storage organelles or plasma membrane channels Second messengers FIGURE 14.14 Major second messengers, some of the proteins that they regu- late, their sources and their disposition. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 607
608 CHAPTER 14 Principles of cell signaling by phosphorylation of its activation loop. Phosphorylation occurs cotranslationally, and the activation loop phosphorylation is required for assembly of the R2C2 tetramer. The PKAs are mostly cytosolic and are also targeted to specific locations by binding or- ganelle-associated scaffolds (A-kinase anchor- ing proteins, or AKAPs). These AKAPs facilitate phosphorylation of membrane proteins includ- ing GPCRs, transporters, and ion channels. AKAPs can also target PKA to other cellular lo- cations including mitochondria, the cytoskele- ton, and the centrosome. AKAPs often harbor binding sites for other regulatory molecules such as phosphoprotein phosphatases and ad- ditional protein kinases, which allows for co- ordination of multiple signaling pathways and integration of their outputs. PKA generally phosphorylates substrates with a primary consensus motif of Arg-Arg- Xaa-Ser-Hydrophobic, placing it in a large group of kinases that recognize basic residues preced- ing the phosphorylation site. PKA regulates pro- teins throughout the cell ranging from ion chan- nels to transcription factors, and its conserved substrate preference frequently permits predic- tion of substrates by sequence analysis. The cAMP response element binding protein CREB is phosphorylated by PKA on Ser 133 and is largely responsible for the impact of cAMP on transcription of numerous genes. Ca2+ signaling serves diverse purposes in all eukaryotic cells Ca2+ is used as a second messenger in all cells, and is, thus, an even more widespread second messenger than cAMP. Many proteins bind Ca2+ with consequent allosteric changes in their en- zymatic activities, subcellular localization, or interaction with other proteins or with lipids. Direct targets of Ca2+ regulation include almost all classes of signaling proteins described in this chapter, numerous metabolic enzymes, ion channels and pumps, and contractile proteins. Most noteworthy may be muscle actomyosin fibers, which are triggered to contract in re- sponse to cytosolic Ca2+ (see 8.21 Myosin-II func- tions in muscle contraction). Although free Ca2+ is found at concentra- tions near 1 mM in most extracellular fluids, in- tracellular Ca2+ concentrations are maintained near 100 nanomolar levels by the combined ac- tion of pumps and transporters that either ex- trude free Ca2+ or sequester it in the endoplasmic reticulum or mitochondria. Ca2+ signaling is ini- tiated when Ca2+-selective channels in the en- doplasmic reticulum or plasma membrane are opened to allow Ca2+ to enter the cytoplasm. The most important entrance channels include electrically gated channels in animal plasma membranes; a Ca2+ channel in the endoplasmic reticulum that is opened by another second mes- senger, inositol 1,4,5-trisphosphate (see below); and an electrically gated channel in the endo- plasmic (sarcoplasmic) reticulum of muscle that opens in response to depolarization of nearby plasma membrane, a process known as excita- tion-contraction coupling (see 2.9 Plasma mem- Key concepts • Ca2+ serves as a second messenger and regulatory molecule in essentially all cells. • Ca2+ acts directly on many target proteins and also regulates the activity of a regulatory protein calmodulin. • The cytosolic concentration of Ca2+ is controlled by organellar sequestration and release. 14.15 [cAMP] Kinase activity as a function of [cAMP] (%) 100 80 60 40 20 2 x 10-9 2 x 10-8 2 x 10-7 10% 90% (R) Regulatory subunits 4 cAMP(C) Catalytic subunits C R R C - cAMP - cAMP - cAMP - cAMP C R R C R2C2 + 4 cAMP R2 . cAMP4 + 2C PKA Activated PKA Activation of PKA by cAMP FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Binding of four molecules of cAMP to the reg- ulatory subunits induces dissociation of two molecules of C, the active form of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel, the cooperative binding of four molecules of cAMP generates a steep acti- vation profile. Activity increases from approximately 10% to 90% as the cAMP concentration increases only 10-fold. An apparent threshold is intro- duced because there is little change in activity at low concentrations of cAMP. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 608
14.16 Lipids and lipid-derived compounds are signaling molecules 609 brane Ca2+ channels activate intracellular functions). In addition to the proteins that are regulated by binding Ca2+ directly, many other proteins re- spond to Ca2+ by binding a widespread Ca2+ sen- sor, the small, ~17 kDa protein calmodulin. Calmodulin requires the binding of four mole- cules of Ca2+ to become fully active, and bind- ing is highly cooperative, generating a sigmoid activation profile illustrated in FIGURE 14.16. Calmodulin generally binds its targets in a Ca2+- dependent manner, but Ca2+-free calmodulin may remain bound but inactive in some cases. For example, calmodulin is a constitutive sub- unit of phosphorylase kinase that is activated upon Ca2+ binding. Higher plants again make major modifications to this paradigm. Calmodulin is not expressed as a distinct protein but, instead, is found as a domain in Ca2+-regulated proteins. In yet another variation, the adenylyl cyclase se- creted by the pathogenic bacterium Bordetellaper- tussis is inactive outside cells but is activated by Ca2+-free calmodulin in animal cells, where its rapid production of cAMP is highly toxic. Lipids and lipid-derived compounds are signaling molecules Signals that originate at the plasma membrane may have soluble regulatory targets in the cy- toplasm or intracellular organelles, but integral plasma membrane proteins are also subject to acute controls. For these targets, lipid second messengers may be primary inputs. Lipids de- rived from membrane phospholipids or other Key concepts • Multiple lipid-derived second messengers are produced in membranes. • Phospholipase Cs release soluble and lipid second messengers in response to diverse inputs. • Channels and transporters are modulated by different lipids in addition to inputs from other sources. • PI 3-kinase synthesizes PIP3 to modulate cell shape and motility. • PLD and PLA2 create other lipid second messengers. 14.16 Calcium-free calmodulin calmodulinfree + 4 Ca2+ (Ca 2+) 4 . calmodulin . active target Calcium-bound calmodulin bound to target peptide of CaMK Ca2+ target Calcium binding causes a conformational change in calmodulin 100 80 60 40 20 3 x 10-8 3 x 10-7 3 x 10-6 10% 90% [Ca2+] Activation of target by calmodulin (%) FIGURE 14.16 Ribbon diagrams represent- ing the crystal structures of calmodulin free of Ca2+ and bound to four Ca2+ ions reveal the huge conformational change that calmodulin undergoes upon Ca2+ binding. Ca2+-calmodulin causes activity changes in target proteins. The bottom panel shows the activation of a target by calmodulin as a function of the intracellular free Ca2+ con- centration. The requirement for binding four Ca2+ ions to induce the conformational transition results in cooperative activation of targets. Activity increases from 10% to 90% as the Ca2+ concentration increases only 10-fold. Structures generated from Protein Data Bank files 1CFD and 1MXE. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 609
610 CHAPTER 14 Principles of cell signaling lipid species play numerous roles in cell signal- ing. Because their analysis has been more dif- ficult than for soluble messengers, many probably remain to be discovered and under- stood. FIGURE 14.17 shows the structure of some of these lipids. Phospholipase Cs (PLCs) are the prototyp- ical lipid signaling enzymes. PLC isoforms cat- alyze the hydrolysis of phospholipids between the 3-sn-hydroxyl and the phosphate group to yield a diacylglycerol and phosphate ester. In animals and fungi, PLCs specific for the substrate 2 3 4 5 6 OH 1 OH OH O O O O O P O O- 2 3 4 5 6 OH 1 OH O O O O O O HO OH OH O P O O- O- O O O O O P O O- O O O O OPO3H- OPO3H- OPO3H- OPO3H- H-O3PO HO 2 3 4 5 6 OH 1 OH OPO3H- Phosphatidic acid (PA) Diacylglycerol (DAG) Phosphatidylinositol (PI) Phosphatidylinositol-3,4,5-trisphosphate (PIP3) Inositol trisphosphate (IP3) Structures of some lipid second messengers FIGURE 14.17 Structures of some lipid second messengers and the common precursor phosphatidylinositol. The acyl side chain structures shown here are the most common for mammalian PI lipids. Much of the PA in cells is derived from PC, and its acyl chains may differ from those shown. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 610
14.16 Lipids and lipid-derived compounds are signaling molecules 611 phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyze PIP2 to form two second messengers: 1,2-sn-diacylglycerol (DAG) and inositol-1,4,5- trisphosphate (IP3). The PLC substrate PIP2 is it- self an important regulatory ligand that modulates the activity of several ion channels, transporters, and enzymes. Thus, PLC alters con- centration of three second messengers; its net effect depends on the net turnover of the sub- strate and products. DAG is probably the best known lipid sec- ond messenger; its hydrophobicity limits it to ac- tion in membranes. DAG activates some isoforms of protein kinase C (PKC), modulates the ac- tivity of several cation channels and activates at least one other protein kinase. DAG can be fur- ther hydrolyzed to release arachidonic acid, which can regulate some ion channels. Arachidonic acid is also the precursor of oxida- tion products, such as prostaglandins and throm- boxanes, which are potent extracellular signaling agents. In addition to DAG, PKCs require inter- action with Ca2+ and an acidic phospholipid, such as phosphatidylserine, to become activated. Thus, activation of PKC requires the coincidence of multiple inputs both to generate DAG and to increase intracellular Ca2+. There are more than a dozen PKCs, classified together according to highly conserved sequences in the catalytic do- main. Three subgroups of PKCs, also identifiable by sequence, share different patterns of regu- lation. Their regulation provides examples of many ways in which other mammalian protein kinases are regulated. The first of these groups, canonical PKCs, are generally soluble or very loosely associated with membranes prior to the appearance of DAG. DAG causes their association with mem- branes and permits activation upon binding of other regulators. The second group of PKCs re- quires similar lipids but not Ca2+, and the third group requires other lipids but neither DAG nor Ca2+ for activation. The N-terminal region of PKCs contains a pseudosubstrate domain, a sequence that re- sembles that of a typical substrate except that the target Ser is replaced with Ala. The pseu- dosubstrate region binds to the active site to in- hibit the kinase. Activators cause the pseudosubstrate domain to flip out of the ac- tive site. PKCs are also activated by proteoly- sis, as are many protein kinases with discrete autoinhibitory domains. Proteases clip a flex- ible hinge region, which results in loss of the regulatory domain and consequent activation of the kinase. PKC is the major receptor for phorbol esters, a class of powerful tumor promoters. Phorbol esters mimic DAG and cause a more massive and prolonged activation than physiological stimuli. This massive stimulation can induce proteolysis of PKC, resulting in downregula- tion, or loss of the kinase. (For a personal de- scription on the discovery of protein kinase C see ) IP3, the second product of the PLC reaction, is a soluble second messenger. The most signif- icant IP3 target is a Ca2+ channel in the endo- plasmic reticulum. IP3 causes this channel to open and release stored Ca2+ into the cytoplasm, thereby rapidly elevating the cytosolic Ca2+ over 100-fold and, in turn, causing the activation of numerous targets of Ca2+ signaling. There are at least six families of PIP2-selec- tive PLC enzymes, defined by their distinct forms of regulation, domain compositions, and over- all sequence conservation. Their catalytic do- mains are all quite similar. The PLC-βs are stimulated primarily by Gαq and Gβγ (to individ- ually varying extents). Several are also modu- lated by phosphorylation. PLC-γ isoforms are stimulated by phosphorylation on Tyr residues, frequently by receptor tyrosine kinases. The PLC-ε isoforms are regulated by small, monomeric G proteins of the Rho family. The regulation of the PLC-δs is still incompletely un- derstood. Two other classes similar to the PLC- δs, PLC-η and -ζ, have also been defined recently. (There is no PLC-α.) In addition to their distinct modes of regulation, all of the PLCs are stimu- lated by Ca2+, and Ca2+ often acts synergisti- cally with other stimulatory inputs. This synergy underlies the intensification and prolongation of Ca2+ signaling observed in many cells. Phospholipases A2 and D (PLA2 and PLD) also hydrolyze glycerol phospholipids in cell membranes to form important signaling com- pounds. PLA2 hydrolyzes the fatty acid at the sn- 2 position of multiple phospholipids to produce the cognate lysophospholipid and the free fatty acid, which is generally unsaturated. The free fatty acid is often arachidonic acid, a precursor of extracellular signals. The biological roles of free lysophospholipids are not understood in detail but have been linked to effects on the structure of the membrane bilayer. PLD catalyzes a reaction much like that of PLC but instead hydrolyzes the phosphodiester on the substituent side of the phosphate group to form 3-sn-phosphatidic acid. Cellular PLDs act on multiple glycerol phospholipid substrates, but phosphatidylcholine is probably the sub- EXP : 14-0001 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 611
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
Cobb ross cell signaling chapter
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Cobb ross cell signaling chapter

  • 1. nucleus lipid droplet Golgi RasGAP Ser338 Tyr341 Ser4 Ser62 Raf Tyr Gab2 PI3K Ser Thr SOS Grb2 Grb2 Ser Thr SOSGrb2 Ser Thr SOS Grb2 Ser Thr SOSGrb2 Ser Thr SOS GTP GDP GDP Ras GDP Ras GTP Ras GTP Ras RasGAP P Tyr Gab2 Tyr580 Tyr542 SHP-2 P Tyr Gab2 Tyr580Tyr542SHP-2 P Tyr Gab2 Tyr580Tyr542SHP-2 Tyr544 Tyr559 Tyr697 Tyr706 Tyr721 Tyr807 Tyr921Tyr974 M-CSFR Tyr706 P P Tyr706 P P Tyr701 Ser727STAT1 P Tyr701 P Ser727STAT1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 Tyr Ser312(307:R) IRS Tyr IL-4R Tyr JAK1 TyrJAK3 Tyr JAK1 Tyr IL-4R Tyr JAK1 Tyr IL-4R Tyr common chain TyrJAK3 Tyr common chain TyrJAK3 Tyr common chain y yy y P y P yJ 3 P y P yJ 3 SOCS1 /JAB P Tyr Ser312(307:R) IRS TyrSTAT6 P Tyr STAT6 P Tyr P Tyr STAT6 P Tyr P Tyr STAT6 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 Tyr Tyr STAT6 Tyr440 IFN R1 Tyr JAK1 Tyr1007 JAK2 Tyr JAK1 Tyr440 IFN R1 Tyr JAK1 Tyr440 IFN R1 SHP-1 SOCS1/JAB Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R IL-6R gp130 Tyr JAK1 TyrTyk2 Tyr JAK1 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R Tyr JAK1 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R TyrTyk2 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R TyrTyk2 Tyr759 Tyr767 Tyr814 Tyr905 Tyr915 IL-6R Tyr JAK1 TyrTyk2Tyr JAK1 TyrTyk2 SOCS3 SHP-1 Tyr580 Tyr542 SHP-2 P Tyr580 P Tyr542 SHP-2 Tyr STAT3 P Tyr STAT3 P Tyr P Tyr STAT3 P Tyr P Tyr STAT3 SOCS3 TyrTyk2Tyr IL-10R IL-10RIL-10R Tyr JAK1 Tyr JAK1 Tyr IL-10R Tyr JAK1 Tyr IL-10R TyrTyk2 Tyr IL-10R TyrTyk2 Tyr IL-10R P PP P TLR4 MD-2 IKK TBK-1 P Ser386 P Ser385 IRF-3 Ser Thr Lys IRAK1 IRAK-M SOCS1 /JAB P Ser P Thr Lys IRAK1 Ubc13 Uev1A Ubc13 Uev1A TRIF/ TICAM-1 TBK-1 IKK Ser386 Ser385 IRF-3 IFN- CAPK IKK Ser176 Ser181 IKK IKK IKK Ser176 Ser181 IKK IKK IKK P Ser176 P Ser181 IKK IKK IKK P Ser176 P Ser181 IKK IKK CAPK SCF TrCP P Thr183 P Tyr185 ERK1 ERK2 P Ser276 Ser529 NF- B p65+p50 P Ser276 P Ser529 NF- B p65+p50 Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B IL-10 IL-6 IL-1 p50 I B TNFTNF P Thr183 P Tyr185 P Thr183 P Tyr185 ERK1 ERK2 Ser338 Tyr341 Ser4 Ser62 Raf Tyr STAT5 GM-CSFR GM-CSFR Tyr GM-CSFR TyrJAK1 Tyr1007 JAK2 P Tyr STAT5 P Tyr P Tyr STAT5 P Tyr P Tyr STAT5 Tyr701 Ser727STAT1 IRF-2 IRF-1 IRF-9 Ser484 Ser485 IRF-7 P Tyr701 P Ser727STAT1 P Tyr STAT2 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 IRF-1IRF-2 IFN- IRF-1 IRF-2 NOSII/iNOS IRF-7 IRF-9 P Ser484 P Ser485 IRF-7 P Ser484 P Ser485 IRF-7 IFN- IFN- pyruvate pyruvate acetyl CoA NAD+ NADH+H+ pyruvate carboxylase pyruvate dehydrogenase PDH kinase pyruvate carrier P P P pyruvate dehydrogenase PDH kinase citrate acyl-CoA carnitine CoASH acylcarnitine fatty acid malonyl CoA carnitine acylcarnitine acyl-CoA CPT I CPT II CACT CoASH acyl CoA synthetase fatty acid synthetase acetyl CoA oxaloacetate CoASH acetyl CoA carboxylase citrate liase malate malate dehydrogenase malic enzyme glycerol 3P TG TG O2 hypoxanthine NADPH xanthine e- NADP+ F H2O2 Cl- LOOH Fe3+ e- .O 2 - HOCl acyl CoA synthetase 27-hydroxyChol LXR 9 r 27-hydroxyChol LXR 9 r LXR LXRLXR R SCAP Site Site-2 protease ? acetyl CoA carboxylase fatty acid synthetase Ser383 Ser389Elk-1 P Ser383 P Ser389 Elk-1 PKA Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 Ser32 Ser36 Lys21 Lys22 I B PKA PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Lys21 Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 P Ser32 P Ser36 Ub Lys21 Ub Lys22 I B PKA Ser276 Ser529 NF- B p65+p50 PKA Ser276 Ser529 NF- B p65+p50 PKA CK II NOSII/iNOS NADPH oxidase xanthine oxidase SOD MPO ATP synthetase ATP ADP O2 H+ e- H+ PP2A ys63TRAF6 SerThr LysIRAK1 ys63TRAF6 SerThr LysIRAK1 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 TAB2 Ser ThrTAB1Ser192Thr184 Thr187TAK1 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187TAK1 TAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 Ser ThrTAB1 Ser192Thr184 Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P Thr Ub LysIRAK1 P Ser P Thr Ub LysIRAK1 P Ser P ThrTAB1 P Ser192 P Thr184 P Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 P Ser P ThrTAB1 P Ser192 P Thr184 P Thr187 P TAK1 PTAB2 Ub Lys63TRAF6 c-fos c-jun AP-1 c-Fos+c-Jun Ser21 Ser32 Ser42 Ser70 Ser113 Ser374 c-Fos Ser63 Ser73 c-Jun P Ser63 P Ser73 c-Jun P Ser21P Ser32 P Ser42 P Ser70 P Ser113 P Ser374 c-Fos P Ser63 P Ser73 c-Jun p53 IL-4 IL-4 0 TRAF2 TRAF1 A20TRAF2 TRAF1 A20 IFN IFN A20 PAFR calpain TRAM TRAMTRAM Tyr580 Tyr542 SHP-2 IL-1ra IL-1ra P Ser386 P Ser385 P Ser386 P Ser385 IRF-3 P Ser386 P Ser385 P Ser386 P Ser385 IRF-3 P Tyr STAT6 P Ser276 P Ser529 P Ser276 P Ser529 NF- B p65+p50 P Ser276 P Ser529 P Ser276 P Ser529 NF- B p65+p50 GM-CSF GM-CSF Tyr STAT3 P Tyr STAT3 -2 P Tyr580 P Tyr542 SHP-2 K PI3K Tyr STAT5 P Tyr STAT5 P Tyr P Tyry P Tyr P Tyry P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr JAK1 P TyrTyk2 SOCS3 P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr759 P Tyr767 P Tyr814 P Tyr905 P Tyr915 IL-6R P Tyr JAK1 P TyrTyk2 SOCS3 Tyr IFN R2 P Tyr440 IFN R1 P Tyr IFN R2 P Tyr JAK1 P Tyr1007 JAK2 SOCS1 /JAB P Tyr440 IFN R1 P Tyr IFN R2 P Tyr JAK1 P Tyr1007 JAK2 SOCS1 /JAB Tyr1007 JAK2 Tyr IFN R2 Tyr1007 JAK2 Tyr IFN R2 y Tyr1007y Tyr1007 P y P Tyr1007 P y P Tyr1007 IL-4R p38MAPK P Ser473 P Thr38 Akt/PKB IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 IRF-9 P Tyr STAT2 P Tyr701 P Ser727STAT1 PIAS3 PIAS3 P Tyr P Tyr STAT3 PIAS3 P Tyr P Tyr STAT3 Tyr701 Ser727 Tyr701 Ser727STAT1 MKP PIAS1 PIAS1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 PIAS1 P Tyr701 P Ser727 P Tyr701 P Ser727STAT1 P TyrJAK1 P TyrJAK3 P Tyr IL-4R P Tyr common chain SOCS1 /JAB P TyrJAK1 P TyrJAK3 P Tyr IL-4R P Tyr common chain SOCS1 /JAB SHP-1 SHIP Tyr Fyn P Tyr Fyn PI3K PI3K Ser473Thr38 Akt/PKB P Tyr P Thr JNK proteasome P Ser21P Ser32 P Ser42 P Ser70 P Ser113 P Ser374 c-Fos PP2B Thr183 Tyr185 Thr183 Tyr185 ERK1 ERK2 MKP LXRRXR CPT1 SREBP1c / bHLH SREBP1c / bHLH SREBP1c / bHLH Tyr chain Tyr Tyr chain Fc RIa chain Tyr518 Tyr519 Syk Tyr518 Tyr519 Syk P Tyr518 P Tyr519 Syk P Tyr771 P Tyr783 P Tyr1254 PLC Tyr771 Tyr783 Tyr1254 PLC PP2APP2B Pi P Tyr580 P Tyr542SHP-2 P Tyr580 P Tyr542 SHP-2 P Tyr Gab2 P Tyr580 P Tyr542SHP-2 P Tyr Gab2 P Tyr580 P Tyr542SHP-2 P Ser63 P Ser73 P Ser63 P Ser73 c-Jun P Ser369 P Thr577 P Ser386 P Ser227RSK P Ser369 P Thr577 P Ser386 Ser227 RSK Ser133 CREB P Ser133 CREB Grb2 P Ser P Thr SOSGrb2 P Ser P Thr SOS ASK P MEKK SEK1/MKK4 SEK2/MKK7 P SEK1/MKK4 P SEK2/MKK7 P TyrThr JNK TyrThr JNK Ser473Thr38 Akt/PKB P Ser473 P Thr38 Akt/PKB Tyr P Ser312(307:R) IRS SOCS3 P Tyr P Thr JNK P Thr183 P Tyr185 ERK1 ERK2 P Ser369 P Ser386 R Src P UbcH5 p50 p60 TICAM-1TICAM-1 TRIF/ TICAM-1 CHAPTER OUTLINE 589 Melanie H. Cobb and Elliott M. Ross The University of Texas Southwestern Medical Center at Dallas Introduction Cellular signaling is primarily chemical Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Receptors are catalysts and amplifiers Ligand binding changes receptor conformation Signals are sorted and integrated in signaling pathways and networks Cellular signaling pathways can be thought of as biochemical logic circuits Scaffolds increase signaling efficiency and enhance spatial organization of signaling Independent, modular domains specify protein-protein interactions Cellular signaling is remarkably adaptive Signaling proteins are frequently expressed as multiple species Activating and deactivating reactions are separate and independently controlled Cellular signaling uses both allostery and covalent modification Second messengers provide readily diffusible pathways for information transfer Ca2+ signaling serves diverse purposes in all eukaryotic cells Lipids and lipid-derived compounds are signaling molecules PI 3-kinase regulates both cell shape and the activation of essential growth and metabolic functions Signaling through ion channel receptors is very fast Nuclear receptors regulate transcription G protein signaling modules are widely used and highly adaptable Heterotrimeric G proteins regulate a wide variety of effectors Heterotrimeric G proteins are controlled by a regulatory GTPase cycle Small, monomeric GTP-binding proteins are multiuse switches Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell Two-component protein phosphorylation systems are signaling relays Pharmacological inhibitors of protein kinases may be used to understand and treat disease Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function The Wnt pathway regulates cell fate during development and other processes in the adult Diverse signaling mechanisms are regulated by protein tyrosine kinases Src family protein kinases cooperate with receptor protein tyrosine kinases MAPKs are central to many signaling pathways Cyclin-dependent protein kinases control the cell cycle Diverse receptors recruit protein tyrosine kinases to the plasma membrane What’s next? Summary References 14.36 14.35 14.34 14.33 14.32 14.31 14.30 14.29 14.28 14.27 14.26 14.25 14.24 14.23 14.22 14.21 14.20 14.19 14.18 14.17 14.16 14.15 14.14 14.13 14.12 14.11 14.10 14.9 14.8 14.7 14.6 14.5 14.4 14.3 14.2 14.1 Principles of cell signaling 14 This image represents about 10% of the map of the known signaling interactions and reactions in the mouse macrophage. Preparing such a map in a computable format is the first step in analyzing a large signaling network. This map was prepared by the group led by Hiroaki Kitano at the Systems Biology Institute, Tokyo, using their CellDesigner program. Map courtesy of Kanae Oda, Yukiko Matsuoka, and Hiroaki Kitano (The Systems Biology Institute). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 589
  • 2. 590 CHAPTER 14 Principles of cell signaling nearby), odors, molecules that regulate growth or differentiation, and proteins on the outside of adjacent cells. A mammalian cell typically expresses about fifty distinct receptors that sense different inputs, and, overall, mammals express several thousand receptors. Despite the diversity of cellular lifestyles and the enormous number of substances sensed by different cells, the general classes of proteins and mechanisms involved in signal transduc- tion are conserved throughout living cells, as shown in FIGURE 14.1. • G protein-coupled receptors, composed of seven membrane-span- ning helices, promote activation of het- erotrimeric GTP-binding proteins called G proteins, which associate with the in- ner face of the plasma membrane and convey signals to multiple intracellular proteins. • Receptor protein kinases are often dimers of single membrane-spanning proteins that phosphorylate their in- tracellular substrates and, thus, change the shape and function of the target pro- teins. These protein kinases frequently contain protein interaction domains that organize complexes of signaling pro- teins on the inner surface of the plasma membrane. • Phosphoprotein phosphatases re- verse the effect of protein kinases by re- moving the phosphoryl groups added by protein kinases. • Other single membrane-spanning en- zymes, such as guanylyl cyclase, have an overall architecture similar to the re- ceptor protein kinases but different en- zymatic activities. Guanylyl cyclase catalyzes the conversion of GTP to 3′:5′- cyclic GMP, which is used to propagate the signal. • Ion channel receptors, although di- verse in detailed structure, are usually oligomers of subunits that each contain several membrane-spanning segments. The subunits change their conforma- tions and relative orientations to per- mit ion flux through a central pore. • Two-component systems may either be membrane spanning or cytosolic. The number of their subunits is also vari- able, but each two-component system contains a histidine kinase domain or subunit that is regulated by a signaling molecule and a response regulator that Introduction All cells, from prokaryotes through plants and animals, sense and react to stimuli in their en- vironments with stereotyped responses that al- low them to survive, adapt, and function in ways appropriate to the needs of the organism. These responses are not simply direct physical or metabolic consequences of changes in the local environment. Rather, cells express arrays of sensing proteins, or receptors, that recognize specific extracellular stimuli. In response to these stimuli, receptors regulate the activities of diverse intracellular regulatory proteins that in turn initiate appropriate responses by the cell. The process of sensing external stimuli and conveying the inherent information to intra- cellular targets is referred to as cellular signal transduction. Cells respond to all sorts of stimuli. Microbes respond to nutrients, toxins, heat, light, and chemical signals secreted by other microbes. Cells in multicellular organisms express recep- tors specific for hormones, neurotransmitters, autocrine and paracrine agents (hormone- like compounds from the secreting cell or cells 14.1 Response regulator Sensor Histidine kinase( ( E1 E2 E1 E2 Hetero- trimeric G protein (GPCR) G protein coupled receptor Trans- membrane scaffold Guanylyl cyclaseReceptor protein kinase Ion channel Two- component complex Transcription factor NUCLEUS Overview of major receptor types in a cell FIGURE 14.1 Receptors form a rather small number of families that share com- mon mechanisms of action and overall similar structures. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 590
  • 3. 14.2 Cellular signaling is primarily chemical 591 contains a phosphorylatable aspartate (Asp) residue. • Some receptors are transmembrane scaffolds that change either the con- formation or oligomerization of their intracellular scaffold domains in re- sponse to extracellular signaling mole- cules, or ligands, and, thus, recruit interacting regulatory proteins to a com- mon site on the membrane. • Nuclear receptors are transcription factors, often heterodimers, that may reside in the cytoplasm until activated by agonists or may be permanently lo- cated in the nucleus. The biochemical processes of signal trans- duction are strikingly similar among cells. Bacteria, fungi, plants, and animals use similar proteins and multiprotein modules to detect and process signals. For example, evolutionar- ily conserved heterotrimeric G proteins and G protein-coupled receptors are found in plants, fungi, and animals. Similarly, 3′:5′ cyclic AMP (cAMP) is an intracellular signaling molecule in bacteria, fungi, and animals; and Ca2+ serves a similar role in all eukaryotes. Protein kinases and phosphoprotein phosphatases are used to regulate enzymes in all cells. Although the basic biochemical components and processes of signal transduction are con- served and reused, they are often used in wildly divergent patterns and for many different phys- iological purposes. For example, cAMP is synthe- sized by distantly related enzymes in bacteria, fungi, and animals, and acts on different pro- teins in each organism; it is a pheromone in some slime molds. Cells often use the same series of signaling proteins to regulate a given process, such as transcription, ion transport, locomotion, and metabolism. Such signaling pathways are as- sembled into signaling networks to allow the cell to coordinate its responses to multiple in- puts with its ongoing functions. It is now pos- sible to discern conserved reaction sequences in and between pathways in signaling networks that are analogous to devices within the circuits of analog computers: amplifiers, logic gates, feedback and feed-forward controls, and mem- ory. This chapter discusses the principles and strategies of cellular signaling first and then dis- cusses the conserved biochemical components and reactions of signaling pathways and how these principles are applied. Cellular signaling is primarily chemical Most signals sensed by cells are chemical, and, when physical signals are sensed, they are gen- erally detected as chemical changes at the level of the receptor. For example, the visual pho- toreceptor rhodopsin is composed of the pro- tein opsin, which binds to a second component, the colored vitamin A derivative cis-retinal (the chromophore). When cis-retinal absorbs a photon, it photoisomerizes to trans-retinal, which is an activating ligand of the opsin pro- tein. (For more on rhodopsin signaling see 14.20 G protein signaling modules are widely used and highly adaptable). Similarly, plants sense red and blue light using the photosensory proteins phy- tochrome and cryptochrome, which detect pho- tons that are absorbed by their tetrapyrrole or flavin chromophores. Cryptochrome homologs are also expressed in animals, where they prob- ably mediate adjustment of the diurnal cycle. A few receptors do respond directly to phys- ical inputs. Pressure-sensing channels, which ex- ist in one form or another in all organisms, mediate responses to pressure or shear by chang- ing their ionic conductance. In mammals, hear- ing is mediated indirectly by a mechanically operated channel in the hair cell of the inner ear. The extracellular domain of a protein called cad- herin is pulled in response to acoustic vibration, generating the force that opens the channel. Cells sense mechanical strain through a number of cell surface proteins, including inte- grins. Integrins provide signals to cells based on their attachment to other cells and to molecu- lar complexes in the external milieu. One major group of physically responsive receptors is made up of channels that sense elec- tric fields. Another interesting group are heat/pain-sensing ion channels; several of these heat-sensitive ion channels also respond to chemical compounds, such as capsaicin, the “hot” lipid irritant in hot peppers. Whether a signal is physical or chemical, the receptor initiates the reactions that change the behavior of the cell. We will discuss how these effects are generated in the rest of the chapter. Key concepts • Cells can detect both chemical and physical signals. • Physical signals are generally converted to chemical signals at the level of the receptor. 14.2 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 591
  • 4. 592 CHAPTER 14 Principles of cell signaling Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Receptors mediate responses to amazingly di- verse extracellular messenger molecules; hence, the cell must express a large number of recep- tor varieties, each able to bind its extracellular ligand. In addition, each receptor must be able to initiate a cellular response. Receptors, thus, contain two functional domains: a ligand- binding domain and an effector domain, which may or may not correspond to definable structural domains within the protein. The separation of ligand-binding and effec- tor functions allows receptors for diverse ligands to produce a limited number of evolutionarily conserved intracellular signals through the ac- tion of a few effector domains. In fact, there are Key concepts • Receptors contain a ligand-binding domain and an effector domain. • Receptor modularity allows a wide variety of signals to use a limited number of regulatory mechanisms. • Cells may express different receptors for the same ligand. • The same ligand may have different effects on the cell depending on the effector domain of its receptor. 14.3 only a limited number of receptor families, which are related by their conserved structures and sig- naling functions (see Figure 14.1). There are several useful correlates to the two-domain nature of receptors. For example, a cell can control its responsiveness to an extra- cellular signal by regulating the synthesis or degradation of a receptor or by regulating the receptor’s activity (see 14.10 Cellular signaling is remarkably adaptive). In addition, the nature of a response is gen- erally determined by the receptor and its effec- tor domain rather than any physicochemical property of the ligand. FIGURE 14.2 illustrates the concept that a ligand may bind to more than one kind of receptor and elicit more than one type of response, or several different ligands may all act identically by binding to function- ally similar receptors. For example, the neuro- transmitter acetylcholine binds to two classes of receptors. Members of one class are ion chan- nels; members of the other regulate G proteins. Similarly, steroid hormones bind both to nu- clear receptors, which bind chromatin and reg- ulate transcription, and to other receptors in the plasma membrane. Conversely, when multiple ligands bind to receptors of the same biochemical class, they generate similar intracellular responses. For ex- ample, it is not uncommon for a cell to express several distinct receptors that stimulate produc- tion of the intracellular signaling molecule cAMP. The effect of the receptor on the cell will also be determined significantly by the biology of the cell and its state at any given time. Ligand binding and effector domains may evolve independently in response to varied se- lective pressures. For example, mammalian and invertebrate rhodopsins transduce their signal through different effector G proteins (Gt and Gq, respectively). Another example is calmod- ulin, a small calcium-binding regulatory pro- tein in animals, which in plants appears as a distinct domain in larger proteins. The receptor’s two-domain nature allows the cell to regulate the binding of ligand and the effect of ligand independently. Covalent modification or allosteric regulation can al- ter ligand-binding affinity, the ability of the lig- and-bound receptor to generate its signal or both. We will discuss these concepts further in 14.13 Cellular signaling uses both allostery and co- valent modification. Receptors can be classified either accord- ing to the ligands they bind or the way in which they signal. Signal output, which is character- Ligand A Ligand A Output 1 Output 2 Output 2 Output 1 Output 1 Ligand B Ligand C LBD1 LBD1 LBD1 LBD2 LBD3 ED1 ED1 ED1ED2 ED2 Receptors have a ligand-binding domain and an effector domain CHIMERIC RECEPTOR FIGURE 14.2 Receptors can be thought of as composed of two functional do- mains, a ligand-binding domain (LBD) and an effector domain (ED). The two- domain property implies that two receptors that respond to different ligands (middle) could initiate the same function by activating similar effector do- mains, or that a cell could express two receptor isoforms (left) that respond to the same ligand with distinct cellular effects mediated by different effector do- mains. It also implies that one can create an artificial chimeric receptor with novel properties. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 592
  • 5. 14.5 Ligand binding changes receptor conformation 593 istic of the effector domain, usually correlates best with overall structure and sequence con- servation. (Receptor families grouped by their functions are the organizational basis of the sec- ond half of this chapter.) However, classifying receptors pharmacologically, according to their specificity for ligands, is particularly useful for understanding the organization of endocrine and neuronal systems and for categorizing the multiple physiological responses to drugs. Expression of a receptor that is not nor- mally expressed in a cell is often sufficient to confer responsiveness to that receptor’s ligand. This responsiveness often occurs because the cell expresses the other components necessary for propagating the intracellular signal from the receptor. The precise nature of the response will reflect the biology of the cell. Experimentally, responsiveness to a compound can be induced by introducing the cDNA that encodes the re- ceptor. For example, mammalian receptors may be expressed in yeast, such that the yeast re- spond visibly to receptor ligands, thus provid- ing a way to screen for new chemicals (drugs) that activate the receptor. Finally, it is possible to create chimeric re- ceptors by fusing the ligand-binding domain from one receptor with the effector domain from a different receptor (Figure 14.2). Such chimeras can mediate novel responses to the ligand. With genetic modification of the ligand- binding domain, receptors can be reengineered to respond to novel ligands. Thus, scientists can manipulate cell functions with nonbiological compounds. Receptors are catalysts and amplifiers Receptors act to accelerate intracellular func- tions and are, thus, functionally analogous to en- zymes or other catalysts. Some receptors, including the protein kinases, protein phos- phatases, and guanylate cyclases, are themselves enzymes and thus classical biochemical cata- lysts. More generally, however, receptors use the relatively small energy of ligand binding to accelerate reactions that are driven by alterna- tive energy sources. For example, receptors that are ion channels catalyze the movement of ions Key concepts • Receptors act by increasing the rates of key regulatory reactions. • Receptors act as molecular amplifiers. 14.4 across membranes, a process driven by the elec- trochemical potential developed by distinct ion pumps. G protein-coupled receptors and other guanine nucleotide exchange factors catalyze the exchange of GDP for GTP on the G protein, an energetically favored process dictated by the cell’s nucleotide energy balance. Transcription factors accelerate the formation of the transcrip- tional initiation complex, but transcription it- self is energetically driven by multiple steps of ATP and dNTP hydrolysis. As catalysts, receptors enhance the rates of reactions. Most signaling involves kinetic rather than thermodynamic regulation; that is, sig- naling events change reaction rates rather than their equilibria (see the next section). Thus, sig- naling is similar to metabolic regulation, in which specific reactions are chosen according to their rates, with thermodynamic driving forces playing only a supportive role. In all signaling reactions, receptors use their catalytic activities to function as molecular am- plifiers. Directly or indirectly, a receptor gener- ates a chemical signal that is huge, both energetically and with respect to the number of molecules recruited by a single receptor. Molecular amplification is a hallmark of recep- tors and many other steps in cellular signaling pathways. Ligand binding changes receptor conformation A central mechanistic question in receptor func- tion is how the binding of a signaling molecule to the ligand-binding domain increases the ac- tivity of the effector domain. The key to this question is that receptors can exist in multiple molecular conformations, some active for sig- naling and others inactive. Ligands shift the conformational equilibrium among these con- formations. The structural changes that occur during the receptor’s inactive-active isomeriza- tion and how ligand binding drives these changes are exciting areas of biophysical re- search. However, the basic concept can be de- scribed simply in terms of coupling the conformational isomerizations of the ligand- binding and effector domains. Key concepts • Receptors can exist in active or inactive conformations. • Ligand binding drives the receptor toward the active conformation. 14.5 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 593
  • 6. 594 CHAPTER 14 Principles of cell signaling How do ligands activate (or not activate) a receptor? Most of the basic regulatory activities of receptors can be described by a simple scheme that considers the receptors as having two in- terconvertible conformations, inactive (R) and active (R*). R and R* are in equilibrium, which is described by the equilibrium constant J. Because unliganded receptors are usually minimally active, J<<1 and an unliganded recep- tor spends most of its time in the R state. When a signaling molecule (L) binds, it drives the re- ceptor toward the active conformation, R*, in which the effector domain is functional. The lig- and-bound receptor thus spends most of its time in the active R* state. The mechanism whereby a ligand can ac- tivate receptor is a simple consequence of its relative affinities for the receptor’s active and inactive conformations. A ligand can bind to the receptor in either of its conformations, de- scribed here by association constants K for the R state and K* for the R* state. Any ligand that binds with higher affinity for the R* conforma- tion than for R will be an activator. If K* is greater than K, the ligand is an agonist. According to the Second Law of Thermodynamics, a system of R + L J R*+ L R L J* K*K R* L R J R* coupled equilibria displays path independence: the net free energy difference between two states is independent of which intermediary re- actions take place. For the receptor, any path from R to R*L therefore has the same free en- ergy change, and the products of the equlib- rium constants along each path are equal. For the example above, path independence means that: J•K* = K•J* Therefore, J* / J = K* / K. Thus, if binding to the R* configuration is preferred (i.e., K*/K>>1), then ligand binding will shift the conformation to the R* state to an equivalent extent (i.e., J*/J>>1). The relative activation by a saturating concentration of lig- and, J*/J, will exactly equal the ligand’s relative selectivity for the active receptor conformation, K*/K. This argument is generally valid for the reg- ulation of a protein’s activity by any regulatory ligand. This model explains many properties of re- ceptors and their ligands both simply and quan- titatively. • First, J must be greater than zero for the equilibrium to exist. Thus, even unli- ganded receptor has some activity. Overexpressed receptors frequently dis- play their intrinsic low activity. • Because physiological receptors are nearly inactive in the absence of ligand, J must be much less than 1 and is prob- ably less than 0.01; most receptors are less than 1% active without agonist. • Ligands can vary in their selectivities between R and R*. Their abilities to ac- tivate will also vary. Some ligands, re- ferred to as agonists, can drive formation of appreciable R*. Others, known as par- tial agonists, will promote submaxi- mal activation. Chemical manipulation of a ligand’s structure will often alter its activity as an agonist. These relation- ships are depicted graphically in FIGURE 14.3. • A ligand that binds equally well to both the R and R* states will not cause acti- vation. However, such a ligand may still occupy the binding site and thereby competitively inhibit binding of an ac- tivating ligand. Such competitive in- hibitors, referred to as antagonists, are frequently used as drugs to block un- wanted activation of a receptor in var- ious disease states. • A ligand that binds preferentially to R 0 1.0 0.8 0.6 0.4 0.2 0 0.012 0.010 0.008 0.006 0.004 0.002 Log [L] Partial agonist Log [L] High affinity agonist Lower affinity agonist Fractional activity of receptor Fractional activity of receptor Inverse agonist Receptor ligands can vary in their activities and potencies FIGURE 14.3 The simple two-state model shown here can describe a wide va- riety of behaviors displayed by receptors and their various regulatory ligands. The left panel shows fractional activity of a receptor exposed to two agonists with different affinities and one partial agonist. The right panel shows the ef- fect of an inverse agonist. If the low fractional activity of unliganded receptor is detected as significant biological activity, then its inhibition by the inverse agonist would be easily detectable. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 594
  • 7. 14.6 Signals are sorted and integrated in signaling pathways and networks 595 relative to R* will further shift the con- formational equilibrium to the inactive state and cause net inhibition. Such lig- ands are called inverse agonists. Because J is already low, effects of in- verse agonists may only be noticeable if a receptor is overexpressed or if the receptor is mutated to increase its in- trinsic activity (i.e., the mutation in- creases J). • The extent to which an agonist stimu- lates a receptor is unrelated to its affin- ity. Both agonists and antagonists may bind with either high or low affinity. Affinity does determine the receptor’s sensitivity—that is, how low a concen- tration of ligand can the receptor detect. Affinities of receptors for natural regu- latory ligands vary enormously, with physiologic Kd values ranging from <10-12 M for some hormones to about 10-3 M for some bacterial chemoattrac- tants. Another aspect of sensitivity is how abruptly or gradually the receptor is activated as the concentration of ag- onist increases. The above model pre- dicts that a receptor is activated significantly at agonist concentrations between 0.1 and 10 times its Kd. A va- riety of cellular mechanisms can con- vert such a conventional response range of about 100-fold to either a more grad- ual response or a very steep, switchlike response. • This model only describes equilibria. It makes no predictions about the rates of ligand binding or release, or of the con- formational isomerization that leads to activation. This model shows how three important as- pects of receptor action are independently de- termined. As mentioned above, affinity for ligand, which determines the concentration range over which the ligand functions, is inde- pendent of the ligand’s net effectiveness at driv- ing receptor activation. The rate of response is also largely independent of these other two properties. Each aspect of receptor function can thus be independently regulated in response to other incoming signals or by the metabolic or developmental state of the cell. Such control of signal input is central to whole-cell coordina- tion of signal transduction. Examples and mech- anisms will recur throughout this chapter. Signals are sorted and integrated in signaling pathways and networks Receptors rarely act directly on the intracellu- lar processes that they ultimately regulate. Rather, receptors typically initiate a sequence of regulatory events that involve intermediary proteins and small molecules. The use of mul- tistep signaling pathways allows cells to amplify signals, adjust signaling kinetics, insert control points, integrate multiple signals, and route sig- nals to distinct effectors. Branched pathways give cells the ability to integrate multiple incoming signals and to di- rect information to the correct control points. As FIGURE 14.4 illustrates, branching can be ei- ther convergent, with multiple signals regulat- ing common end points, or divergent, with a single pathway branching to control more than one process. In multicellular organisms, diver- gent branching allows a single hormone recep- tor to initiate distinct cell-appropriate patterns of responses in different cells and tissues. Divergent signaling also allows a receptor to regulate qualitatively different cellular responses with quantitatively distinct intensities, each de- pendent on signal amplification in the interme- diary pathway. Convergent branching—when several re- ceptors activate the same pathway to elicit the same regulatory responses—is also common. Convergent branching allows multiple incom- ing signals, both stimulatory and inhibitory, to be integrated and coordinately regulated at a common site downstream of the receptors. Receptors for several different hormones fre- quently initiate similar or overlapping patterns of signaling in a single target cell. Overlapping converging and diverging sig- naling pathways create signaling networks within cells that coordinate responses to multiple in- puts (Figure 14.4). Typically, such pathways are complex in the number and diversity of their components and in the topology of their circuit Key concepts • Signaling pathways usually have multiple steps and can diverge and/or converge. • Divergence allows multiple responses to a single signal. • Convergence allows signal integration and coordination. 14.6 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 595
  • 8. 596 CHAPTER 14 Principles of cell signaling maps. Signaling networks are also spatially com- plex. They may include components in various subcellular locations, with initial receptors and associated proteins in the plasma membrane, but with downstream proteins in the cytoplasm or in- tracellular organelles. Such complexity is neces- sary to allow the cells to integrate and sort incoming signals and to regulate multiple intra- cellular functions simultaneously. The complexity and adaptability of signal- ing networks, like the one shown in the lower half of Figure 14.4, make their dynamics at the whole-cell level difficult or impossible to grasp intuitively. Signaling networks resemble large analog computers, and investigators are increas- ingly depending on computational tools to un- derstand cellular information flow and its regulation. First, many signaling interactions that include only two or three proteins exert functions analogous to traditional computa- tional logic circuits (see the next section). The theory and experience with such circuits in elec- tronics facilitate understanding biological sig- naling functions as well. The enormous complexity of cellular signal- ing networks can be simplified by considering them to be composed of interacting signaling modules, i.e., groups of proteins that process sig- nals in well-understood ways. A cellular signal- ing module is analogous to an integrated circuit in an electronic instrument that performs a known function, but whose exact components could be changed for similar use in another de- vice. The concept of modular construction facil- itates both qualitative and quantitative understanding of signaling networks. We will re- fer to many standard signaling modules later in the chapter. Examples include monomeric and heterotrimeric G protein modules, MAPK cas- cades, tyrosine (Tyr) kinase receptors and their binding proteins, and Ca2+ release/uptake mod- ules. In each case, despite the numerous phylo- genetic, developmental, and physiologic variations, understanding the basic function of that class of module conveys understanding of all its incarnations. Last, the evolutionary impor- tance of modules is significant; once the architec- ture of a module is established it can be reused. For larger-scale networks, multiplexed, high-throughput measurements on living cells have been combined with powerful kinetic mod- eling strategies to allow an increasingly accurate quantitative depiction of information flow within signaling modules or entire networks. Such models, with sound and experimentally based parameter sets, can describe signaling processes in systems too complex for intuitive or ad hoc analysis. They are also vital as tests of understanding because they can predict exper- imental results in ways that can be used to test the validity of the model. Well-grounded mod- els can then be used (cautiously) to suggest the mechanisms of systems for which data sets re- main unattainable. At even greater levels of complexity, the theories and tools of computer science are increasingly giving useful systems- level analyses of signal flow in cells. Using com- putational tools to analyze large arrays of quantitative data allows us to understand cel- lular information flow and its regulation. Linear, parallel Convergent Divergent Multiply branched RECEPTORS TRANSDUCERS EFFECTORS Convergent and divergent signaling pathways FIGURE 14.4 Signaling pathways use convergent and divergent branching to co- ordinate information flow. The diagrams at top show how even a simple, three- level signaling network can sort information. Convergence or divergence can take place at multiple points along a signaling pathway. As an example of com- plexity, the lower portion of the figure shows a small segment (~10%) of the G protein-mediated signaling network in a mouse macrophage cell line. It omits several interpathway regulatory mechanisms and completely ignores inputs from non-G protein-coupled receptors. Pathway map courtesy of Lily Jiang, University of Texas Southwestern Medical Center. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 596
  • 9. 14.7 Cellular signaling pathways can be thought of as biochemical logic circuits 597 Developing quantitative models of signaling networks is a frontier in signaling biology. These models both help describe network function and pinpoint experiments to clarify mechanism. Cellular signaling pathways can be thought of as biochemical logic circuits As introduced in the preceding section, processes thatsignalingpathwaysusetointegrateanddirect information to cellular targets are strikingly anal- ogoustothemathematicallogicfunctionsthatare used to design the individual circuits of electronic computers. Indeed, there are biological equiva- lents of essentially all of the functional compo- nents that computer scientists and engineers considerinthedesignofcomputersandelectronic control devices. To understand signaling path- ways, it is, therefore, useful to consider groups of reactionswithinapathwayasconstitutinglogiccir- cuits of the sort used in electronic computing, as illustrated in FIGURE 14.5. The simplest example is whentwostimulatorypathwaysconverge.Ifsuf- ficient input from either is adequate to elicit the response, the convergence would constitute an “OR” function. If neither input is sufficient by it- self but the combination of the two elicits the re- sponse, then the converging pathways would create “AND” functions. AND circuits are also re- ferredtoascoincidencedetectors—aresponse is elicited only when two stimulating pathways are activated simultaneously. AND functions can result from the combi- nation of two similar but quantitatively inade- quate inputs. Alternatively, two mechanistically different inputs might both be required to elicit a response. An example of the latter would be a target protein that is allosterically activated only when phosphorylated, or that is activated by phosphorylation but is only functional when recruited to a specific subcellular location. The opposite of an AND circuit is a NOT function, where one pathway blocks the stim- Key concepts • Signaling networks are composed of groups of biochemical reactions that function as mathematical logic functions to integrate information. • Combinations of such logic functions combine as signaling networks to process information at more complex levels. 14.7 ulatory effect of another. Simple logic gates are observed at many locations in cellular signaling pathways. We can also think about convergent signal- ing in quantitative rather than Boolean terms by considering the additivity of inputs to a dis- tinct process (see Figure 14.5, right). The OR function referred to above can be considered to be the additive positive inputs of two pathways. Such additivity could represent the ability of several receptors to stimulate a pool of a partic- ular G protein or the ability of two protein ki- nases to phosphorylate a single substrate. Additivity may be positive, as in the examples above, or negative, such as when two inhibitory inputs combine. Inhibition and stimulation may also combine additively to yield an algebraically balanced output. Alternatively, multiple inputs can combine with either more or less than an additive effect. The NOT function, discussed above, is analogous to describing a blockade of stimulation. The AND function describes syn- ergism, where one input potentiates another but alone has little effect. Even simple signaling networks can display complex patterns of information processing. One Additive Logical (Boolean) Quantitative (Analog) Response A + B Response A OR B A NOT B A AND B B A Response Response B A A + fixed [B] A + B A Response Response A + B B A Less than additive More than additive A + B Response B A A + B B A Response log (agonist concentration) log (agonist concentration) log (agonist concentration) B Simple logic circuits FIGURE 14.5 Signaling networks use simple logic functions to process information. Boolean OR, AND, and NOT functions (left) correspond to the quantitative interactions between converging signals that are shown on the right. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 597
  • 10. 598 CHAPTER 14 Principles of cell signaling good example is the creation of “memory”: mak- ing the effect of a transient signal more or less permanent. Signaling pathways have multiple ways of setting memories, and of forgetting. One mechanism, common in protein kinase path- ways, is the positive feedback loop, illustrated in the top panel of FIGURE 14.6. In a positive feed- back loop, the input stimulates a transducer (T), which in turn stimulates the effector protein (E) to create the output. If the effector can also ac- tivate the transducer, sufficient initial signal can be fed back to the transducer that it can main- tain the effector's full signal output even when input is removed. Such systems typically display a threshold behavior, as shown on the right. A positive feed-forward loop can generate memory of another type (Figure 14.6, middle panel), indicating the duration of input. In such circuits, the effector requires simultaneous in- put from both the receptor and from the inter- mediary transducer. If the pathway from receptor through transducer is relatively slow, or if it requires the accumulation of a substan- tial amount of transducer, only a prolonged in- put will trigger a response, as shown in the time-base output diagram at the right. A third way to establish memory is to allow one input to control the reversibility of a sec- ond regulatory event (Figure 14.6, bottom panel). WASP, a protein that initiates the polymerization of actin to drive cellular motion and shape change, is activated both by phosphorylation and by the binding of Cdc42, a small GTP-bind- ing protein (G). However, the phosphorylation site on WASP is only exposed when WASP is bound to Cdc42. Phosphorylation thus requires both activated Cdc42 and activated protein ki- nase. If Cdc42 dissociates, the phosphorylated state of WASP persists until another signaling molecule, whose identity remains uncertain, binds again to expose the site to a protein phos- phatase. As shown in the time-base graph, ex- posure to Cdc42 will activate, but exposure to kinase alone will not. If Cdc42 is present, then the kinase can activate WASP. Phospho-WASP is relatively insensitive to protein phosphatase (P) alone, but can be dephosphorylated if Cdc42 or another G protein binds to expose the site to phosphatase. Scaffolds increase signaling efficiency and enhance spatial organization of signaling Key concepts • Scaffolds organize groups of signaling proteins and may create pathway specificity by sequestering components that have multiple partners. • Scaffolds increase the local concentration of signaling proteins. • Scaffolds localize signaling pathways to sites of action. 14.8 Positive feedback loop : irreversible ON switch Positive feed-forward loop : responds to prolonged input Conformational lock - Dual control switch Input Input strength Output Output T Input OutputT Output Time Time Output + + input Kinase Phosphatase OH G OH G P G P G P G K PG K G P E E OH E E E E E E Signal processing circuits FIGURE 14.6 Relatively complex signal processing can be executed by simple multi-protein modules. The figure depicts three types of signaling modules (left) and their behavior in response to agonist (right). (top) In a positive feed-back module, a transducer protein (T) stimulates an effector (E) to pro- duce a cellular output, but the effector also stimulates the activity of the trans- ducer. The result can be an all-or-none switch, where input up to a threshold has little effect, but then becomes committed when feedback from the effec- tor is sufficient to maintain transducer activity even in the absence of contin- ued input from the receptor. (center) In a positive feed-forward module, the effector requires input both from the transducer and from upstream in the path- way. When stimulation is brief (short horizontal bar under trace at right), sig- nificant amounts of active transducer do not accumulate and output is minimal. When stimulation is prolonged (longer bar), signal output is substantial. (bot- tom) In some dual-control switching modules, the binding of one regulator (G) can both activate the effector and expose another regulatory site, shown here as a Ser substrate site (-OH) for a protein kinase. The effector can only be phos- phorylated or dephosphorylated when G is bound. Therefore, as shown at the right, addition of G alone will activate but activation of the kinase (K) alone will not. If kinase is active while G is bound, phosphorylation is resistant to phosphatase activity unless G is again present to reexpose the phosphoserine residue (shown on the graph at the right as a bold P). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 598
  • 11. 14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling 599 The proteins in a signaling pathway are fre- quently colocalized within cells such that their mutual interactions are favored and their in- teractions with other proteins are minimized. Many signaling pathways are organized on scaf- folds. Scaffolds bind several components of a signaling pathway in multiprotein complexes to enhance signaling efficiency. Scaffolds pro- mote interactions of proteins that have a low affinity for each other, accelerate activation (and often inactivation) of the associated compo- nents, and localize the signaling proteins to ap- propriate sites of action. Colocalization may be tonic or regulated, and stimulus-dependent scaf- folding often determines signaling outputs. The binding sites on a scaffolding protein are often localized in distinct modular protein- binding domains, giving the impression that the protein is designed simply to hold the compo- nents of the pathway together. Many scaffold- ing proteins do lack intrinsic enzymatic activity, but some signaling enzymes also act as scaffolds. Binding to a scaffold facilitates signaling by increasing the local concentrations of the com- ponents, so that diffusion or transport of mol- ecules to their sites of action is not necessary. In the photoreceptor cells of Drosophila, scaffold- ing of signaling components is critical for rapid signal transmission. These cells contain the InaD scaffolding protein, which has five modular binding domains, known as PDZ domains. Each of its PDZ domains binds to a C-terminal motif of a target protein, thereby facilitating interac- tions among the associated proteins. FIGURE 14.7 shows a model for how InaD organizes the sig- naling proteins. The mutational loss of InaD produces a nearly blind fly, and deletion of a single PDZ domain can yield a fly with a dis- tinct visual defect characteristic of the protein that binds to the missing domain. A second example is Ste5p, a scaffold for the pheromone-induced mating response pathway in S. cerevisiae. FIGURE 14.8 illustrates how Ste5p binds and organizes components of a mitogen- PKC CYTOSOL TRP Rhodopsin CaMCaM PDZ PDZ - PDZ PKC PDZ - PDZ INAD PDZZ INAD PDZPDZ PDZ PDZ PDZ The INAD signaling complex FIGURE 14.7 The scaffold InaD organizes proteins that transmit visual signals in the fly photoreceptor cell. InaD is localized to the photorecep- tor membrane and coordinates light sensing and visual transduction. In invertebrate eyes, the visual signaling pathway goes from rhodopsin through Gq to a phospholipase C-␤, and Ca2+ release triggered by PLC ac- tion initiates depolarization. This system is specialized for speed, and re- quires that the relevant proteins are nearby. InaD contains five PDZ domains, each of which binds to the C terminus of a signal transducing protein. The TRP channel, which mediates Ca2+ entry, PLC-␤, and a pro- tein kinase C isoform that is involved in rapid desensitization all bind con- stitutively to InaD. Rhodopsin and a myosin (NinaC) also bind, and Gq binds indirectly. Scaffold determines specificity of Ste11p signaling Scaffold organizes MAPK cascade Ste11p Ste11p Ste20p Ste7p Ste7p Fus3p Fus3p Ste20p Ste20pSte20p Ste20pSte20p Ste7p Fus3p Ste11p Pheromone Cdc42pCdc42p Cdc42pCdc42p Mating response G protein Ste5p Ste5p Pbs2p Pheromone High osmolarity Osmo- adaptation Mating response Ste11p Hog1p Scaffolds concentrate and insulate signaling proteins GPCR FIGURE 14.8 The scaffold Ste5p organizes the components of the MAPK cascade that mediates the pheromone-induced mating response in Saccharomyces cerevisiae. In the top left panel, Ste5p brings the compo- nents of the MAPK cascade to the membrane in response to pheromone. In the top right panel, binding to the heterotrimeric G protein brings loaded Ste5p in proximity to the protein kinase Ste20p bound to the activated small GTP binding protein Cdc42p. Their colocalization facilitates the sequential activation of the cascade components, resulting in activation of the MAPK Fus3p and the mating response. The MAP3K Ste11p can regulate not only the MAPK Fus3p in the mating pathway, but also the MAPK Hog1p in the high osmolarity pathway, as shown in the bottom two panels. The scaffold to which Ste11p binds, either Ste5p or Pbs2 (both a scaffold and a MAP2K), determines which MAPK and downstream events are activated as the out- put. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 599
  • 12. 600 CHAPTER 14 Principles of cell signaling activated protein kinase (MAPK) cascade, in- cluding a MAP3K (Ste11p), a MAP2K (Ste7p) and a MAPK (Fus3p). (The MAPK cascade will be discussed in 14.32 MAPKs are central to many signaling pathways). The function of Ste5p is par- tially retained even if the positions of its bind- ing sites for the kinases are shuffled in the linear sequence of the protein, indicating that a major role is to bring the enzymes into proximity, rather than to precisely orient them. Ste5p also binds to the ␤␥ subunits of the heterotrimeric G pro- tein that mediates the actions of mating pheromones, linking the membrane signal to the intracellular transducers. Yeast that lack Ste5p cannot mate, demonstrating that Ste5p is required for this biological function (but not all functions) carried out by the pathway. In addition to facilitating signaling in their own pathways, scaffolds can enhance signaling specificity by limiting interactions with other signaling proteins. Scaffolds thus insulate com- ponents of a signaling pathway both from acti- vation by inappropriate signals and from producing incorrect outputs. For example, the mating and osmosensing pathways in yeast share several components, including the MAP3K Ste11p, but each pathway maintains specificity because it employs different scaffolds that restrict signal transmission. In contrast, the presence of excess scaffold can inhibit signaling because the individual sig- naling components will more frequently bind to distinct scaffold proteins rather than forming a functional complex. Such dilution among scaf- folds causes separation rather than concentra- tion of the components, preventing their productive interaction. Independent, modular domains specify protein- protein interactions Modular protein interaction domains or motifs occur in many signaling proteins and confer the ability to bind structural motifs in other mole- cules, including proteins, lipids, and nucleic Key concepts • Protein interactions may be mediated by small, conserved domains. • Modular interaction domains are essential for signal transmission. • Adaptors consist exclusively of binding domains or motifs. 14.9 acids. Some of these domains are listed in FIGURE 14.9. In contrast to scaffolds, which bind spe- cific proteins with considerable selectivity, mod- ular interaction domains generally recognize not a single molecule but a group of targets that share related structural features. Modular interaction domains important for signal transduction were first discovered in the protein tyrosine kinase proto-oncogene Src, which contains a protein tyrosine kinase do- main and two domains named Src homology (SH) 2 and 3 domains. The modular SH2 and SH3 domains were originally identified by com- parison of Src to two other tyrosine kinases, Fps and Abl. One or both of these domains appear in numerous proteins and both are critically in- volved in protein-protein interactions. SH3 domains, which consist of approxi- mately 50 residues, bind to specific short pro- line-rich sequences. Many cytoskeletal proteins and proteins found in focal adhesion complexes contain SH3 domains and proline rich se- quences, suggesting that this targeting motif may send proteins with these domains to these sites of action within cells. In contrast to phos- photyrosine-SH2 binding, the proline-rich bind- ing sites for SH3 domains are present in resting and activated cells. However, SH3-proline inter- actions may be negatively regulated by phospho- rylation within the proline-rich motif. SH2 domains, which consist of approxi- mately 100 residues, bind to Tyr phosphory- lated proteins, such as cytoplasmic tyrosine kinases and receptor tyrosine kinases. Thus, Tyr phosphorylation regulates the appearance of SH2 binding sites and, thereby, regulates a set of protein-protein interactions in a stimulus- dependent manner. A clever strategy was used to identify the binding specificity of SH2 domains. An isolated recombinant SH2 domain was incubated with cell lysates and then recovered from the lysates using a purification tag. The proteins associated with the SH2 domain were some of the same proteins that were recognized by antiphospho- tyrosine antibodies. By this and other methods, it was discovered that SH2 domains recognize sequences surrounding Tyr phosphorylation sites and require phosphorylation of the in- cluded Tyr for high affinity binding. Information on specific amino acid se- quences that recognize and bind to modular binding domains is being accumulated as these individual interactions are identified. In addi- tion, screening programs using cDNA and/or peptide libraries to assess binding capabilities 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 600
  • 13. 14.9 Independent, modular domains specify protein-protein interactions 601 Characteristics of some common modular protein domains 14-3-3 Binds protein phosphoserine or phosphothreonine Protein sequestration Domain Characteristics Cellular involvement WW Binds proline-rich sequences Alternative to SH3; vesicular trafficking TPR Degenerate sequence of ~34 amino acids with residues WL/GYAFAP; forms a scaffold Wide variety of processes SH3 Binds to PXXP motifs Various processes SH2 Binds to protein phosphotyrosine (pY) Tyrosine protein kinase signaling SAM Homo- and hetero- oligomerization Wide variety of processes RING Binds zinc and may be found in E3 ubiquitin ligases Ubiquitination, transcription PH Binds to specific phosphoinositi- des, esp. PI-4,5-P2, PI-3,4-P2 or PI-3,4,5-P3. Recruitment to mem- branes and motility PDZ Binds to the C-terminal 4-5 residues of proteins that have a hydrophobic residue at the terminus; may bind to PIP2 Scaffolding diverse protein complexes often at the membrane LIM Zinc-binding cysteine-rich motif that forms two tandemly repeated zinc fingers Wide variety of processes HECT Binds E2 ubiquitin-conjugating enzymes to transfer ubiquitin to the substrate or to ubiquitin chains Ubiquitination FYVE Binds to PI(3)P Membrane trafficking, TGF-␤ signaling FHA Binds protein phosphothreonine or phosphoserine Various; DNA damage F-Box Binds Skp1 in a ubiquitin-ligase complex Ubiquitination EF hand Binds calcium Calcium-dependent processes C2 Binds phospholipids Signal transduction, vesicular trafficking C1 Binds phorbol esters or diacyl- glycerol Recruitment to mem- branes Dimerization Caspase activation Bromo CARD Binds acetylated lysine residues Chromatin-associated proteins FIGURE 14.9 The table describes a subset of known modular protein in- teraction domains found in many proteins. Interactions mediated by these domains are essential to controlling cell function. Few if any of these do- mains exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/). yield such motifs. Consensus target sequences for individual domains have been identified based on the sequence specificity of their bind- ing to arrayed sequences. These consensus se- quences can then be used to predict whether the domain will bind a site in a candidate pro- tein. Adaptor proteins, which lack enzymatic activity, link signaling molecules and target them in a manner that is responsive to extra- cellular signals. Adaptor proteins are generally made up of two or more modular interaction domains or the complementary recognition motifs. Unlike scaffolds, adaptors are usually 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 601
  • 14. 602 CHAPTER 14 Principles of cell signaling multifunctional because their modular interac- tion domains and motifs are not as highly spe- cific. Adaptors bind to two or more other signaling proteins via their protein-protein in- teraction domains to colocalize them or to fa- cilitate additional interactions. Grb2 is a prototypical adaptor protein that was identified as a protein that bound to the C- terminal region of the EGF receptor. Grb2 has one SH2 and two SH3 domains. It binds consti- tutively to specific proline-rich segments of pro- teins through its SH3 domain, although this binding can be negatively regulated. One target of Grb2 is SOS, a guanine nucleotide exchange factor that activates the small GTP-binding pro- tein Ras in response to EGF signaling. Through its SH2 domain, Grb2 binds Tyr-phosphorylated proteins, including the receptors themselves in a stimulus-dependent manner. Thus, Tyr phos- phorylation of these receptors in response to ligand will enable the binding of Grb2 to the re- ceptors, which, in turn, will recruit SOS to the membrane-localized receptor. Once at the mem- brane, SOS can activate its target, Ras. Cellular signaling is remarkably adaptive Auniversalpropertyofcellularsignalingpathways is adaptation to the incoming signal. Cells contin- uously adjust their sensitivity to signals to main- taintheirabilitytodetectchangesininput.Typically, when a cell is exposed to a new input, it initiates a process of desensitization that dampens the cel- lularresponsetoanewplateaulowerthantheini- tial peak response, as illustrated in FIGURE 14.10. When the stimulus is removed, the desensitized state can persist, with sensitivity slowly returning to normal. Similarly, the removal of a tonic stim- ulus can hypersensitize signaling systems. Key concepts • Sensitivity of signaling pathways is regulated to allow responses to change over a wide range of signal strengths. • Feedback mechanisms execute this function in all signaling pathways. • Most pathways contain multiple adaptive feedback loops to cope with signals of various strengths and durations. 14.10 Initial response Heterologous desensitization Homologous desensitization Time Time R1 R2 R2 X1 X2 Y Z Response R esponse a b K a R1 R2 Z X1 X2 Agonist Agonist Agonist Desensitization Agonist a for R1 Reapply a or b Agonist a for R1 Reapply a or b Time Response R1R1 R2R1 or a Y Patterns of adaptation in signaling networksFIGURE 14.10 Top: Upon exposure to a stimulus, signaling pathways adjust their sensitivities to adapt to the new level of input. Thus, the response de- cays after initial stimulation. A sec- ond similar stimulus will elicit a smaller response unless adequate time is al- lowed for recovery. Bottom: Some adap- tation mechanisms feed back only on the receptor that is stimulated and do not alter parallel pathways. Such mech- anisms are referred to as homologous. At left, agonist a for receptor R1 can initiate either of two feedback events that desensitize R1 alone. In other cases, a stimulus will also cause par- allel or related systems to desensitize. At the right, agonist a initiates desen- sitization of both R1 and R2. The re- sponse to agonist b, which binds to R2, is also desensitized. Such heterol- ogous desensitization is common. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 602
  • 15. 14.10 Cellular signaling is remarkably adaptive 603 Adaptation in signaling is one of the best ex- amples of biological homeostasis. The adaptabil- ity of cellular signaling can be quite impressive. Cells commonly regulate their sensitivity to phys- iological stimuli over more than a 100-fold range, and the mammalian visual response can adapt to incoming light over a 107-fold range. This re- markable ability allows a photoreceptor cell to detect a single photon, and allows a person to read in both very dim light and intense sunlight. Adaptability is observed in bacteria, plants, fungi, and animals. Many of its properties are conserved throughout biology, although the most complex adaptive mechanisms are found in animals. The general mechanism for adaptation is the nega- tive feedback loop, which biochemically samples the signal and controls the adaptive process. Adaptationvarieswithboththeintensityand the duration of the incoming signal. Stronger or more persistent inputs tend to drive greater adap- tive change and, often, adaptation that persists for a longer time. Cells can modulate adaptation in this way because adaptation is exerted by a successionofindependentmechanisms,eachwith its own sensitivity and kinetic parameters. G protein pathways offer excellent examples of adaptation. FIGURE 14.11 shows that the earli- est step in adaptation is receptor phosphoryla- tion, which is catalyzed by G protein-coupled receptor kinases (GRKs) that selectively recog- nize the receptor’s ligand-activated conforma- tion.Phosphorylationinhibitsthereceptor’sability to stimulate G protein activation and also pro- motes binding of arrestin, a protein that further inhibits G protein activation. Moreover, arrestin binding primes receptors for endocytosis, which removes them from the cell surface. Endocytosis can also be the first step in receptor proteolysis. Along with these direct effects, many receptor genes display feedback inhibition of transcrip- tion, such that signaling by a receptor decreases its own expression. Stimulation thus causes multiple adaptive processes that range from immediate (phospho- rylation, arrestin binding) through delayed (tran- scriptionalregulation),andincludebothreversible and irreversible events. This array of adaptive events has been demonstrated for many G pro- tein-coupled receptors, and many cells may use all of them to control output from one receptor. The speed, extent, and reversibility of adaptation are selected by a cell’s developmental program. Cells can change their patterns of adaptation both qualitatively and quantitatively by altering the points in a pathway where feedback is initi- ated and exerted. In a linear pathway, changing DNA GRK Relative response Agonist added Endosomal receptor degradation Receptor transcription inhibited Receptor phosphorylation Arrestin binding Receptor endocytosis G protein Early endosome Lysosome Time (seconds) 0 1 10 100 1000 Agonist binds G protein active EFFECTORS Arrestin GPCR G P C R gene 1 2 3 5 4 5 Receptor transcription inhibited C Y T O P L A S M N U C L E U S Agonist Receptor degradation 4 Receptor endocytosis 3 Receptor recycling 1 Receptor phosphorylation Arrestin binding 2 Multiple adaptation processes occur after a stimulus FIGURE 14.11 Multiple adaptation processes are invoked during a stimulus, and multiple nested mechanisms for adaptation are the rule. They are usually invoked sequentially according to the duration and intensity of the stimulus. For GPCRs, at least five desensitizing mechanisms are known, with others act- ing on the G protein and effectors. these points will alter the kinetics or extent of adaptation(Figure14.10).Inbranchedpathways, changing these points can determine whether adaptation is unique to one input or is exerted formanysimilarinputs.Ifreceptoractivationtrig- gers its desensitization directly, or if an event downstream on an unbranched pathway triggers desensitization,thenonlysignalsthatinitiatewith that receptor will be altered. Receptor-selective adaptation is referred to as homologous adap- tation (Figure 14.10). 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 603
  • 16. 604 CHAPTER 14 Principles of cell signaling Alternatively, feedback control can initiate downstream from multiple receptors in a con- vergent pathway and thus regulate both the initiating receptor and the others. Such het- erologous adaptation regulates all the possi- ble inputs to a given control point. A common example is the phosphorylation of G protein- coupled receptors by either protein kinase A or protein kinase C, which are activated by down- stream signals cAMP or Ca2+ plus the lipid dia- cylglycerol, respectively. Like GRK, these kinases both attenuate receptor activity and promote arrestin binding. Cells also alter their responses to incoming signals for homeostatic reasons. These consid- erations include phase of the cell cycle, meta- bolic status, or other aspects of cellular activity. Again, all these adaptive processes may be dis- played to a greater or lesser extent in different cells, different pathways within a cell or differ- ent situations during the cell’s lifetime. Signaling proteins are frequently expressed as multiple species Key concepts • Distinct species (isoforms) of similar signaling proteins expand the regulatory mechanisms possible in signaling pathways. • Isoforms may differ in function, susceptibility to regulation or expression. • Cells may express one or several isoforms to fulfill their signaling needs. 14.11 Cells increase the richness, adaptability, and regulation of their signaling pathways by ex- pressing multiple species of individual signal- ing proteins that display distinct biochemical properties. These species may be encoded by multiple genes or by multiple mRNAs derived from a single gene by alternative splicing or mRNA editing. The numerical complexity im- plicit in these choices is impressive. Consider the neurotransmitter serotonin: In mammals, there are thirteen serotonin receptors, each of which stimulates a distinct spectrum of G pro- teins of the Gi, Gs, and Gq families. (A four- teenth serotonin receptor is an ion channel.) FIGURE 14.12 shows the relationship of serotonin receptors to these G protein families. There is also tremendous diversity among the G proteins and adenylyl cyclases. There are three genes for Gαi and one each for the closely related Gαz and Gαo. Furthermore, the Gαo mRNA is multiply spliced. There are four Gq members. In addition, there are five genes for Gβ and twelve for Gγ, and most of the possible Gβγ dimers are expressed naturally. There are ten genes for adenylyl cyclases, which are direct targets of Gs and either direct or indirect targets of the other G proteins. While all nine mem- brane-bound adenylyl cyclase isoforms are stim- ulated by Gαs, they display diverse stimulatory and inhibitory responses to Gβγ, Gαi, Ca2+, calmodulin, and several protein kinases, as il- lustrated in FIGURE 14.13. Thus, stimulation by serotonin can lead to diverse responses depend- ing upon the various forms of the proteins that are engaged at a particular time and location. FIGURE 14.12 Receptors for serotonin have evolved in mammals as a family of 13 genes that regulate three of the four major classes of G pro- teins. While all respond to the natural ligand serotonin, the binding sites have evolved suf- ficient differences that drugs have been devel- oped that specifically target one or more isoforms. The type 3 serotonin receptors, not shown here, are ligand-gated ion channels and are not obviously related to the others. 1B Gi Gs Gs Gq 1D 1E 1F 1A 7 5A 5B 4 2A 2C 2B 6 120 100 80 60 40 20 0 Isoforms Nucleotide substitution distance G protein Evolutionary relationship of serotonin receptor isoforms 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 604
  • 17. 14.12 Activating and deactivating reactions are separate and independently controlled 605 Sometimes isoforms of a signaling protein are subject to quite different kinds of inputs. For example, all of the members of the phospho- lipase C family (PLC) hydrolyze phosphatidyli- nositol-4,5-bisphosphate to form two second messengers, diacylglycerol and inositol-1,4,5 trisphosphate (see 14.16 Lipids and lipid-derived compounds are signaling molecules). The distinct isoforms may be regulated by diverse combina- tions of Gαq, Gβγ, phosphorylation, monomeric G proteins, or Ca2+. Because a cell has multiple options when expressing a form of a signaling protein, it can use expression of particular isoforms to alter how it performs otherwise identical signaling functions. Different cells express one or more isoforms to allow appropriate responses, and ex- pression can vary according to other inputs or the cell’s metabolic status. In addition, signaling pathways are remarkably resistant to mutational or other injuries because loss of a single species or isoform of a signaling protein can often be compensated for by increased expression or ac- tivity of another species. Similarly, engineered overexpression can result in the reduced expres- sion of endogenous proteins. The existence of multiple receptor species can, thus, substantially add to adaptability and the consequent resist- ance of signaling networks to damage. Activating and deactivating reactions are separate and independently controlled In signaling networks, individual proteins are frequently activated and deactivated by distinct reactions, a feature that facilitates separate reg- ulation. Common examples include using pro- tein kinases and phosphoprotein phosphatases Key concepts • Activating and deactivating reactions are usually executed by different regulatory proteins. • Separating activation and inactivation allows for fine-tuned regulation of amplitude and timing. 14.12 CaM CaMK Gαs Gαi Gβγ PKA PKC inhibit activate Regulators Ca2+ NO Different isoforms of adenylyl cyclase are regulated differently FIGURE 14.13 All of the mammalian membrane-bound adenylyl cyclases are structurally homologous and catalyze the same reaction, and all are stimulated by G␣s. Their responses to other inputs (protein kinases CaMK, PKA and PKC; Ca2+; calmodulin (CaM); NO•) are specific to each isoform, allowing a rich com- binatoric input to cellular cAMP signaling. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 605
  • 18. 606 CHAPTER 14 Principles of cell signaling to catalyze protein phosphorylation and de- phosphorylation; using adenylyl cyclase to cre- ate cAMP while using phosphodiesterases to hydrolyze it or anion transporters to pump it out of the cell; or using GTP/GDP exchange fac- tors (GEFs) to activate G proteins and GTPase- activating proteins (GAPs) to deactivate them. Depending on stoichiometry and detailed mech- anism, these strategies can convey either addi- tive or nonadditive inputs while maintaining fine control over the kinetics of activation and deactivation of a signaling pathway. The use of distinct reactions for activation and deactiva- tion is analogous to the use of distinct anabolic and catabolic enzymes in reversible metabolic pathways. Cellular signaling uses both allostery and covalent modification Cellular signaling uses almost every imagina- ble mechanism for regulating the activities of intracellular proteins, but most can be described as either allosteric or covalent. Individual sig- naling proteins typically respond to multiple al- losteric and covalent inputs. Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. Because a protein’s activity reflects its confor- mation, the binding of any molecule that alters conformation can change the target protein’s activity. Any molecule can have allosteric ef- fects: protons or Ca2+, small organic molecules, or other proteins. Allosteric regulation can be both inhibitory or stimulatory. Covalent modification of a protein’s chem- ical structure is also frequently used to regulate its activity. The change in the protein’s chemi- cal structure alters its conformation and, thus, its activity. Most regulatory covalent modifica- tion is reversible. The classic and most common regulatory covalent event is phosphorylation, in which a phosphoryl group is transferred from ATP to the protein, most often to the hydroxyl group of serine (Ser), threonine(Thr), or tyro- sine (Tyr). Enzymes that phosphorylate proteins Key concepts • Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. • Modification of a protein’s chemical structure is also frequently used to regulate its activity. 14.13 are known as protein kinases. Their actions are opposed by phosphoprotein phosphatases, which catalyze the hydrolysis of the phosphoryl group to yield free phosphate and restore the unmod- ified hydroxyl residue. Other forms of covalent modification are also common and will be ad- dressed throughout the chapter. Second messengers provide readily diffusible pathways for information transfer Signaling pathways make use of both proteins and small molecules according to their distinc- tive attributes. A small molecule used as an in- tracellular signal, or second messenger, has a number of advantages over a protein as a sig- naling intermediary. Small molecules can be synthesized and destroyed quickly. Because they can be made readily, they can act at high con- centrations so that their affinities for target pro- teins can be low. Low affinity permits rapid dissociation, such that their signals can be ter- minated promptly when free second messenger molecules are destroyed or sequestered. Because second messengers are small, they also can dif- fuse quickly within the cell, although many cells have developed mechanisms to spatially restrict such diffusion. Second messengers are, thus, superior to proteins in mediating fast responses, particularly at a distance. Second messengers are also useful when signals have to be addressed to large numbers of target proteins simultane- ously. These advantages often overcome their lack of catalytic activity and their inability to bind multiple molecules simultaneously. FIGURE 14.14 lists intracellular second mes- sengers developed through evolution. This num- ber is surprisingly low. Several are nucleotides synthesized from major metabolic nucleotide precursors. They include cAMP, cyclic GMP, ppGppp, and cyclic ADP-ribose. Other soluble second messengers include a sugar phosphate, inositol-1,4,5-trisphosphate(IP3),adivalentmetal ion Ca2+, and a free radical gas nitric oxide (NO•). Lipid second messengers include diacylglycerol and phosphatidylinositol-3,4,5-trisphosphate, Key concepts • Second messengers can propagate signals between proteins that are at a distance. • cAMP and Ca2+ are widely used second messengers. 14.14 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 606
  • 19. 14.14 Second messengers provide readily diffusible pathways for information transfer 607 phosphatidylinositol-4,5-diphosphate, sphingo- sine-1-phosphate and phosphatidic acid. The first signaling compound to be described as a second messenger was cAMP. The name arose because cAMP is synthesized in animal cells as a second, intracellular signal in response to numerous extracellular hormones, the first messengers in the pathway. cAMP is used by prokaryotes, fungi, and animals to convey in- formation to a variety of regulatory proteins. (Its occurrence in higher plants has still not been proved.) Adenylyl cyclases, the enzymes that syn- thesize cAMP from ATP, are regulated in vari- ous ways depending on the organism in which they occur. In animals, adenylyl cyclase is an integral protein of the plasma membrane whose multiple isoforms are stimulated by diverse agents (see Figure 14.13). In animal cells, adeny- lyl cyclase is generally stimulated by Gs, which was originally discovered as an adenylyl cyclase regulator. Some fungal adenylyl cyclases are also stimulated by G proteins. Bacterial cyclases are far more diverse in their regulation. cAMP is removed from cells in two ways. It may be extruded from cells by an ATP-driven anion pump but is more often hydrolyzed to 5′- AMP by members of the cyclic nucleotide phos- phodiesterase family, a large group of proteins that are themselves under multiple regulatory controls. The prototypical downstream regulator for cAMP in animals is the cAMP-dependent pro- tein kinase, but a bacterial cAMP-regulated tran- scription factor was discovered shortly thereafter, and other effectors are now known (Figure 14.14). The cAMP system remains the proto- typical eukaryotic signaling pathway in that its components exemplify almost all of the recog- nized varieties of signaling molecules and their interactions: hormone, receptor, G protein, adenylyl cyclase, protein kinase, phosphodi- esterase, and extrusion pump. The second messenger-stimulated protein kinase PKA is a tetramer composed of two cat- alytic (C) subunits and two regulatory (R) sub- units, as illustrated in FIGURE 14.15. The R subunit binds to the catalytic subunit in the substrate- binding region, maintaining C in an inhibited state. Each R subunit binds two molecules of cAMP, four cAMP molecules per PKA holoen- zyme. When these sites are filled, the R subunit dimer dissociates rapidly, leaving two free cat- alytic subunits with high activity. The difference in affinity of R for C in the presence and absence of cAMP is ~10,000-fold. The strongly cooper- ative binding of cAMP generates a very steep activation curve with an apparent threshold be- low which no significant activation of PKA oc- curs, as illustrated in Figure 14.15. PKA activity, thus, increases dramatically over a narrow range of cAMP concentrations. PKA is also regulated Protein kinase A Bacterial trans- cription factors Cation channel Cyclic nucleotide phospho- diesterase Rap GDP/GTP exchange factor (Epac) RNA polymerase ObgE trans- cription arrest detector IP3-gated Ca2+ channel Protein kinase C Trp cation channel Ion channel Transporters Protein kinase G Cation channel Cyclic nucleotide phospho- diesterase Ca2+ channel Various two component system proteins Guanylyl cyclase Numerous calmodulin Akt (protein kinase B) Other PH domains/proteins Adenylyl cyclase Rel1A SpoT Phospho- lipase C Phospho- lipase C PIP 5-kinase PI 3-kinase Guanylyl cyclase ADP-ribose cyclase Diguanylate cyclase NO. synthase ATP GTP PIP2 PIP2 PI-4-P GTP NAD GTP arginine Stored Ca2+ PIP2 Phospho- diesterase Organic anion transporter SpoT- catalyzed hydrolysis Phosphatase Diacylglycerol kinase Diacylglycerol lipase Phospho- lipase C Phosphatase Phospho- diesterase Hydrolysis Cyclic di-GMP phospho- diesterase Reduction Reuptake and extrusion pumps Phosphatase Magic spot 3':5'-cyclic AMP (cAMP) (ppGpp, ppGppp) Inositol-1,3,5- trisphosphate (IP3) Diacylglycerol (DAG) Phosphatidyl- inositol-4,5- bisphosphate (PIP2) 3':5'-Cyclic GMP (cGMP) Cyclic ADP-ribose Cyclic diguanosine- monophosphate Nitric oxide (NO.) Ca2+ Phosphatidyl- inositol-3,4,5- trisphosphate Second messenger Synthesis/ ReleaseTargets Pre- cursor Removal Release from storage organelles or plasma membrane channels Second messengers FIGURE 14.14 Major second messengers, some of the proteins that they regu- late, their sources and their disposition. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 607
  • 20. 608 CHAPTER 14 Principles of cell signaling by phosphorylation of its activation loop. Phosphorylation occurs cotranslationally, and the activation loop phosphorylation is required for assembly of the R2C2 tetramer. The PKAs are mostly cytosolic and are also targeted to specific locations by binding or- ganelle-associated scaffolds (A-kinase anchor- ing proteins, or AKAPs). These AKAPs facilitate phosphorylation of membrane proteins includ- ing GPCRs, transporters, and ion channels. AKAPs can also target PKA to other cellular lo- cations including mitochondria, the cytoskele- ton, and the centrosome. AKAPs often harbor binding sites for other regulatory molecules such as phosphoprotein phosphatases and ad- ditional protein kinases, which allows for co- ordination of multiple signaling pathways and integration of their outputs. PKA generally phosphorylates substrates with a primary consensus motif of Arg-Arg- Xaa-Ser-Hydrophobic, placing it in a large group of kinases that recognize basic residues preced- ing the phosphorylation site. PKA regulates pro- teins throughout the cell ranging from ion chan- nels to transcription factors, and its conserved substrate preference frequently permits predic- tion of substrates by sequence analysis. The cAMP response element binding protein CREB is phosphorylated by PKA on Ser 133 and is largely responsible for the impact of cAMP on transcription of numerous genes. Ca2+ signaling serves diverse purposes in all eukaryotic cells Ca2+ is used as a second messenger in all cells, and is, thus, an even more widespread second messenger than cAMP. Many proteins bind Ca2+ with consequent allosteric changes in their en- zymatic activities, subcellular localization, or interaction with other proteins or with lipids. Direct targets of Ca2+ regulation include almost all classes of signaling proteins described in this chapter, numerous metabolic enzymes, ion channels and pumps, and contractile proteins. Most noteworthy may be muscle actomyosin fibers, which are triggered to contract in re- sponse to cytosolic Ca2+ (see 8.21 Myosin-II func- tions in muscle contraction). Although free Ca2+ is found at concentra- tions near 1 mM in most extracellular fluids, in- tracellular Ca2+ concentrations are maintained near 100 nanomolar levels by the combined ac- tion of pumps and transporters that either ex- trude free Ca2+ or sequester it in the endoplasmic reticulum or mitochondria. Ca2+ signaling is ini- tiated when Ca2+-selective channels in the en- doplasmic reticulum or plasma membrane are opened to allow Ca2+ to enter the cytoplasm. The most important entrance channels include electrically gated channels in animal plasma membranes; a Ca2+ channel in the endoplasmic reticulum that is opened by another second mes- senger, inositol 1,4,5-trisphosphate (see below); and an electrically gated channel in the endo- plasmic (sarcoplasmic) reticulum of muscle that opens in response to depolarization of nearby plasma membrane, a process known as excita- tion-contraction coupling (see 2.9 Plasma mem- Key concepts • Ca2+ serves as a second messenger and regulatory molecule in essentially all cells. • Ca2+ acts directly on many target proteins and also regulates the activity of a regulatory protein calmodulin. • The cytosolic concentration of Ca2+ is controlled by organellar sequestration and release. 14.15 [cAMP] Kinase activity as a function of [cAMP] (%) 100 80 60 40 20 2 x 10-9 2 x 10-8 2 x 10-7 10% 90% (R) Regulatory subunits 4 cAMP(C) Catalytic subunits C R R C - cAMP - cAMP - cAMP - cAMP C R R C R2C2 + 4 cAMP R2 . cAMP4 + 2C PKA Activated PKA Activation of PKA by cAMP FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Binding of four molecules of cAMP to the reg- ulatory subunits induces dissociation of two molecules of C, the active form of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel, the cooperative binding of four molecules of cAMP generates a steep acti- vation profile. Activity increases from approximately 10% to 90% as the cAMP concentration increases only 10-fold. An apparent threshold is intro- duced because there is little change in activity at low concentrations of cAMP. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 608
  • 21. 14.16 Lipids and lipid-derived compounds are signaling molecules 609 brane Ca2+ channels activate intracellular functions). In addition to the proteins that are regulated by binding Ca2+ directly, many other proteins re- spond to Ca2+ by binding a widespread Ca2+ sen- sor, the small, ~17 kDa protein calmodulin. Calmodulin requires the binding of four mole- cules of Ca2+ to become fully active, and bind- ing is highly cooperative, generating a sigmoid activation profile illustrated in FIGURE 14.16. Calmodulin generally binds its targets in a Ca2+- dependent manner, but Ca2+-free calmodulin may remain bound but inactive in some cases. For example, calmodulin is a constitutive sub- unit of phosphorylase kinase that is activated upon Ca2+ binding. Higher plants again make major modifications to this paradigm. Calmodulin is not expressed as a distinct protein but, instead, is found as a domain in Ca2+-regulated proteins. In yet another variation, the adenylyl cyclase se- creted by the pathogenic bacterium Bordetellaper- tussis is inactive outside cells but is activated by Ca2+-free calmodulin in animal cells, where its rapid production of cAMP is highly toxic. Lipids and lipid-derived compounds are signaling molecules Signals that originate at the plasma membrane may have soluble regulatory targets in the cy- toplasm or intracellular organelles, but integral plasma membrane proteins are also subject to acute controls. For these targets, lipid second messengers may be primary inputs. Lipids de- rived from membrane phospholipids or other Key concepts • Multiple lipid-derived second messengers are produced in membranes. • Phospholipase Cs release soluble and lipid second messengers in response to diverse inputs. • Channels and transporters are modulated by different lipids in addition to inputs from other sources. • PI 3-kinase synthesizes PIP3 to modulate cell shape and motility. • PLD and PLA2 create other lipid second messengers. 14.16 Calcium-free calmodulin calmodulinfree + 4 Ca2+ (Ca 2+) 4 . calmodulin . active target Calcium-bound calmodulin bound to target peptide of CaMK Ca2+ target Calcium binding causes a conformational change in calmodulin 100 80 60 40 20 3 x 10-8 3 x 10-7 3 x 10-6 10% 90% [Ca2+] Activation of target by calmodulin (%) FIGURE 14.16 Ribbon diagrams represent- ing the crystal structures of calmodulin free of Ca2+ and bound to four Ca2+ ions reveal the huge conformational change that calmodulin undergoes upon Ca2+ binding. Ca2+-calmodulin causes activity changes in target proteins. The bottom panel shows the activation of a target by calmodulin as a function of the intracellular free Ca2+ con- centration. The requirement for binding four Ca2+ ions to induce the conformational transition results in cooperative activation of targets. Activity increases from 10% to 90% as the Ca2+ concentration increases only 10-fold. Structures generated from Protein Data Bank files 1CFD and 1MXE. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 609
  • 22. 610 CHAPTER 14 Principles of cell signaling lipid species play numerous roles in cell signal- ing. Because their analysis has been more dif- ficult than for soluble messengers, many probably remain to be discovered and under- stood. FIGURE 14.17 shows the structure of some of these lipids. Phospholipase Cs (PLCs) are the prototyp- ical lipid signaling enzymes. PLC isoforms cat- alyze the hydrolysis of phospholipids between the 3-sn-hydroxyl and the phosphate group to yield a diacylglycerol and phosphate ester. In animals and fungi, PLCs specific for the substrate 2 3 4 5 6 OH 1 OH OH O O O O O P O O- 2 3 4 5 6 OH 1 OH O O O O O O HO OH OH O P O O- O- O O O O O P O O- O O O O OPO3H- OPO3H- OPO3H- OPO3H- H-O3PO HO 2 3 4 5 6 OH 1 OH OPO3H- Phosphatidic acid (PA) Diacylglycerol (DAG) Phosphatidylinositol (PI) Phosphatidylinositol-3,4,5-trisphosphate (PIP3) Inositol trisphosphate (IP3) Structures of some lipid second messengers FIGURE 14.17 Structures of some lipid second messengers and the common precursor phosphatidylinositol. The acyl side chain structures shown here are the most common for mammalian PI lipids. Much of the PA in cells is derived from PC, and its acyl chains may differ from those shown. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 610
  • 23. 14.16 Lipids and lipid-derived compounds are signaling molecules 611 phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyze PIP2 to form two second messengers: 1,2-sn-diacylglycerol (DAG) and inositol-1,4,5- trisphosphate (IP3). The PLC substrate PIP2 is it- self an important regulatory ligand that modulates the activity of several ion channels, transporters, and enzymes. Thus, PLC alters con- centration of three second messengers; its net effect depends on the net turnover of the sub- strate and products. DAG is probably the best known lipid sec- ond messenger; its hydrophobicity limits it to ac- tion in membranes. DAG activates some isoforms of protein kinase C (PKC), modulates the ac- tivity of several cation channels and activates at least one other protein kinase. DAG can be fur- ther hydrolyzed to release arachidonic acid, which can regulate some ion channels. Arachidonic acid is also the precursor of oxida- tion products, such as prostaglandins and throm- boxanes, which are potent extracellular signaling agents. In addition to DAG, PKCs require inter- action with Ca2+ and an acidic phospholipid, such as phosphatidylserine, to become activated. Thus, activation of PKC requires the coincidence of multiple inputs both to generate DAG and to increase intracellular Ca2+. There are more than a dozen PKCs, classified together according to highly conserved sequences in the catalytic do- main. Three subgroups of PKCs, also identifiable by sequence, share different patterns of regu- lation. Their regulation provides examples of many ways in which other mammalian protein kinases are regulated. The first of these groups, canonical PKCs, are generally soluble or very loosely associated with membranes prior to the appearance of DAG. DAG causes their association with mem- branes and permits activation upon binding of other regulators. The second group of PKCs re- quires similar lipids but not Ca2+, and the third group requires other lipids but neither DAG nor Ca2+ for activation. The N-terminal region of PKCs contains a pseudosubstrate domain, a sequence that re- sembles that of a typical substrate except that the target Ser is replaced with Ala. The pseu- dosubstrate region binds to the active site to in- hibit the kinase. Activators cause the pseudosubstrate domain to flip out of the ac- tive site. PKCs are also activated by proteoly- sis, as are many protein kinases with discrete autoinhibitory domains. Proteases clip a flex- ible hinge region, which results in loss of the regulatory domain and consequent activation of the kinase. PKC is the major receptor for phorbol esters, a class of powerful tumor promoters. Phorbol esters mimic DAG and cause a more massive and prolonged activation than physiological stimuli. This massive stimulation can induce proteolysis of PKC, resulting in downregula- tion, or loss of the kinase. (For a personal de- scription on the discovery of protein kinase C see ) IP3, the second product of the PLC reaction, is a soluble second messenger. The most signif- icant IP3 target is a Ca2+ channel in the endo- plasmic reticulum. IP3 causes this channel to open and release stored Ca2+ into the cytoplasm, thereby rapidly elevating the cytosolic Ca2+ over 100-fold and, in turn, causing the activation of numerous targets of Ca2+ signaling. There are at least six families of PIP2-selec- tive PLC enzymes, defined by their distinct forms of regulation, domain compositions, and over- all sequence conservation. Their catalytic do- mains are all quite similar. The PLC-βs are stimulated primarily by Gαq and Gβγ (to individ- ually varying extents). Several are also modu- lated by phosphorylation. PLC-γ isoforms are stimulated by phosphorylation on Tyr residues, frequently by receptor tyrosine kinases. The PLC-ε isoforms are regulated by small, monomeric G proteins of the Rho family. The regulation of the PLC-δs is still incompletely un- derstood. Two other classes similar to the PLC- δs, PLC-η and -ζ, have also been defined recently. (There is no PLC-α.) In addition to their distinct modes of regulation, all of the PLCs are stimu- lated by Ca2+, and Ca2+ often acts synergisti- cally with other stimulatory inputs. This synergy underlies the intensification and prolongation of Ca2+ signaling observed in many cells. Phospholipases A2 and D (PLA2 and PLD) also hydrolyze glycerol phospholipids in cell membranes to form important signaling com- pounds. PLA2 hydrolyzes the fatty acid at the sn- 2 position of multiple phospholipids to produce the cognate lysophospholipid and the free fatty acid, which is generally unsaturated. The free fatty acid is often arachidonic acid, a precursor of extracellular signals. The biological roles of free lysophospholipids are not understood in detail but have been linked to effects on the structure of the membrane bilayer. PLD catalyzes a reaction much like that of PLC but instead hydrolyzes the phosphodiester on the substituent side of the phosphate group to form 3-sn-phosphatidic acid. Cellular PLDs act on multiple glycerol phospholipid substrates, but phosphatidylcholine is probably the sub- EXP : 14-0001 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 611