Protein phosphorylation via protein kinases and dephosphorylation via protein phosphatases regulates many cellular processes through signal transduction cascades. G-protein coupled receptors (GPCRs) detect extracellular signals and activate intracellular heterotrimeric G proteins, which then modulate the activity of effector proteins like adenylate cyclase. When activated by GTP, Gα subunits dissociate from Gβγ complexes to control downstream signaling events. Effector activation is terminated upon GTP hydrolysis by Gα, allowing it to reassociate with Gβγ.

1. Signal Transduction
Mahesh D. Mahendrakar
Lecturer in biotechnology
Biochemistry of Metabolism

2. Many enzymes are regulated by covalent
attachment of phosphate, in ester linkage, to
the side-chain hydroxyl group of a particular
amino acid residue (serine, threonine, or
tyrosine).
threonine (Thr)serine (Ser)

3.  A protein kinase transfers the terminal phosphate of ATP
to a hydroxyl group on a protein.
 A protein phosphatase catalyzes removal of the Pi by
hydrolysis.
Protein OH + ATP Protein O P
O
O−
O−
+ ADP
Pi H2O
Protein Kinase
Protein Phosphatase

4. Phosphorylation may directly alter activity of an
enzyme, e.g., by promoting a conformational change.
Alternatively, altered activity may result from
binding another protein that specifically recognizes
a phosphorylated domain.
 E.g., 14-3-3 proteins bind to domains that include
phosphorylated Ser or Thr in the sequence
RXXX[pS/pT]XP, where X can be different amino acids.
 Binding to 14-3-3 is a mechanism by which some proteins
(e.g., transcription factors) may be retained in the cytosol,
& prevented from entering the nucleus.

5. Protein kinases and phosphatases are themselves
regulated by complex signal cascades. For example:
 Some protein kinases are activated by Ca++
-calmodulin.
 Protein Kinase A is activated by cyclic-AMP (cAMP).
Protein OH + ATP Protein O P
O
O−
O−
+ ADP
Pi H2O
Protein Kinase
Protein Phosphatase

6. Adenylate Cyclase (Adenylyl
Cyclase) catalyzes:
ATP  cAMP + PPi
Binding of certain hormones
(e.g., epinephrine) to the outer
surface of a cell activates
Adenylate Cyclase to form
cAMP within the cell.
Cyclic AMP is thus considered
to be a second messenger.
N
N N
N
NH2
O
OHO
HH
H
H2
C
H
O
P
O
O-
1'
3'
5' 4'
2'
cAMP

7. Phosphodiesterase enzymes
catalyze:
cAMP + H2O  AMP
The phosphodiesterase that
cleaves cAMP is activated by
phosphorylation catalyzed by
Protein Kinase A.
Thus cAMP stimulates its
own degradation, leading to
rapid turnoff of a cAMP signal.
N
N N
N
NH2
O
OHO
HH
H
H2
C
H
O
P
O
O-
1'
3'
5' 4'
2'
cAMP

8. Protein Kinase A (cAMP-Dependent Protein
Kinase) transfers Pi from ATP to OH of a Ser or Thr
in a particular 5-amino acid sequence.
Protein Kinase A in the resting state is a complex of:
• 2 catalytic subunits (C)
• 2 regulatory subunits (R).
R2C2

9. R2C2
Each regulatory subunit (R) of Protein Kinase A
contains a pseudosubstrate sequence, like the
substrate domain of a target protein but with
Ala substituting for the Ser/Thr.
The pseudosubstrate domain of (R), which
lacks a hydroxyl that can be phosphorylated,
binds to the active site of (C), blocking its
activity.

10. R2C2 + 4 cAMP  R2cAMP4 + 2C
When each (R) binds 2 cAMP, a conformational
change causes (R) to release (C).
The catalytic subunits can then catalyze
phosphorylation of Ser or Thr on target proteins.
PKIs, Protein Kinase Inhibitors, modulate activity
of the catalytic subunits (C).
View an animation of activation of Protein Kinase
A.

11. Rhodopsin PDB 1F88
G Protein Signal Cascade
Most signal molecules targeted to a cell bind at the cell
surface to receptors embedded in the plasma membrane.
Only signal molecules able to cross
the plasma membrane (e.g., steroid
hormones) interact with intracellular
receptors.
A large family of cell surface
receptors have a common structural
motif, 7 transmembrane α-helices.
Rhodopsin was the first of these to
have its 7-helix structure confirmed
by X-ray crystallography.

12.  Rhodopsin is unique.
It senses light, via a bound
chromophore, retinal.
 Most 7-helix receptors have
domains facing the extracellular
side of the plasma membrane that
recognize & bind signal
molecules (ligands).
E.g., the β-adrenergic receptor
is activated by epinephrine &
norepinephrine.
Crystallization of this receptor was aided by genetically
engineering insertion of the soluble enzyme lysozyme
into a cytosolic loop between transmembrane α-helices.
β -Adrenergic
Receptor
PDB 2RH1
Lysozyme
insert
ligand →

13. See an animated image of the β2-adrenergic receptor
structure in a website of the Kobilka lab.
The signal is usually passed from a 7-helix receptor to
an intracellular G-protein.
 Seven-helix receptors are thus called GPCR, or
G-Protein-Coupled Receptors.
 Approx. 800 different GPCRs are encoded in the
human genome.

14. G-protein-Coupled Receptors may dimerize or form
oligomeric complexes within the membrane.
Ligand binding may promote oligomerization, which
may in turn affect activity of the receptor.
Various GPCR-interacting proteins (GIPs) modulate
receptor function. Effects of GIPs may include:
 altered ligand affinity
 receptor dimerization or oligomerization
 control of receptor localization, including transfer to
or removal from the plasma membrane
 promoting close association with other signal proteins

15.  G-proteins are heterotrimeric, with 3 subunits α, β,
γ.
 A G-protein that activates cyclic-AMP formation
within a cell is called a stimulatory G-protein,
designated Gs with alpha subunit Gsα.
 Gs is activated, e.g., by receptors for the hormones
epinephrine and glucagon.
The β-adrenergic receptor is the GPCR for
epinephrine.

16. α & γ subunits have covalently attached lipid anchors that
bind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with
cytosolic domains forming the catalytic site.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
α γ γ + α cytosol
GDP β β GTP
The α subunit of
a G-protein (Gα)
binds GTP, &
can hydrolyze it
to GDP + Pi.

17. The sequence of events by which a hormone
activates cAMP signaling:
1. Initially Gα has bound GDP, and α, β, & γ
subunits are complexed together.
Gβ,γ, the complex of β & γ subunits, inhibits Gα.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
α γ γ + α cytosol
GDP β β GTP

18. 2. Hormone binding, usually to an extracellular domain
of a 7-helix receptor (GPCR), causes a
conformational change in the receptor that is
transmitted to a G-protein on the cytosolic side of
the membrane.
The nucleotide-binding site on Gα becomes more
accessible to the cytosol, where [GTP] > [GDP].
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
α γ γ + α cytosol
GDP β β GTP

19. 3. Substitution of GTP for GDP causes another
conformational change in Gα.
Gα-GTP dissociates from the inhibitory βγ complex &
can now bind to and activate Adenylate Cyclase.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
α γ γ + α cytosol
GDP β β GTP

20. 4. Adenylate Cyclase, activated by the stimulatory
Gα-GTP, catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein
Kinase) catalyzes transfer of phosphate from ATP to
serine or threonine residues of various cellular
proteins, altering their activity.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
α γ γ + α cytosol
GDP β β GTP

21. Turn off of the signal:
1. Gα hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Gα causes it to rebind to the
inhibitory βγ complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterases catalyze hydrolysis of
cAMP  AMP.

22. 3. Receptor desensitization varies with the hormone.
• In some cases the activated receptor is phosphorylated via
a G-protein Receptor Kinase.
• The phosphorylated receptor then may bind to a protein
β-arrestin.
• β-Arrestin promotes removal of the receptor from the
membrane by clathrin-mediated endocytosis.
• β-Arrestin may also bind a cytosolic Phosphodiesterase,
bringing this enzyme close to where cAMP is being produced,
contributing to signal turnoff.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to
proteins via Protein
Kinase A.

23. Signal amplification is an important feature of
signal cascades:
 One hormone molecule can lead to formation of many
cAMP molecules.
 Each catalytic subunit of Protein Kinase A catalyzes
phosphorylation of many proteins during the life-time
of the cAMP.
View an animation of a G-protein signal cascade.

24.  Different isoforms of Gα have different signal roles. E.g.:
 The stimulatory Gsα, when it binds GTP, activates
Adenylate cyclase.
 An inhibitory Giα, when it binds GTP, inhibits
Adenylate cyclase.
Different effectors & their receptors induce Giα to
exchange GDP for GTP than those that activate Gsα.
 The complex of Gβ,γ that is released when Gα binds GTP is itself
an effector that binds to and activates or inhibits several
other proteins.
E.g., Gβ,γ inhibits one of several isoforms of Adenylate
Cyclase, contributing to rapid signal turnoff in cells that
express that enzyme.

25.  Cholera toxin catalyzes covalent modification of Gsα.
• ADP-ribose is transferred from NAD+
to an arginine residue
at the GTPase active site of Gsα.
• ADP-ribosylation prevents GTP hydrolysis by Gsα .
• The stimulatory G-protein is permanently activated.
 Pertussis toxin (whooping cough disease) catalyzes
ADP-ribosylation at a cysteine residue of the inhibitory
Giα, making it incapable of exchanging GDP for
GTP.
• The inhibitory pathway is blocked.
 ADP-ribosylation is a general mechanism by which
activity of many proteins is regulated, in eukaryotes
(including mammals) as well as in prokaryotes.

26. CH2
HH
OH OH
H H
OOP
O
HH
OH OH
H H
O
CH2
N
N
N
NH2
OP
O
−
O
N−
O
(CH2)3
NH
C NH2
+
protein
NH
O
H
C
NH2
O
CH2
H
N
H
OH OH
H H
OOP
O
HH
OH OH
H H
OCH2
N
N
N
NH2
OP
O
O
−
O
N−
O
H
C
NH2
O
N
H
+
+
(CH2)3
NH
C NH2
+
protein
NH2
NAD+
nicotinamide
Arg
residue
ADP-ribosylated
protein
(nicotinamide
adenine
dinucleotide)
ADP
ribosylation

27. These domains include residues adjacent to the
terminal phosphate of GTP and/or the Mg++
associated with the two terminal phosphates.
Inhibitory Gα
GTPγS
PDB 1GIAStructure of G proteins:
The nucleotide binding site
in Gα consists of loops that
extend out from the edge of
a 6-stranded β-sheet.
Three switch domains have
been identified, that change
position when GTP
substitutes for GDP on Gα.

28. GTP hydrolysis occurs by nucleophilic attack of a
water molecule on the terminal phosphate of GTP.
Switch domain II of Gα includes a conserved
glutamine residue that helps to position the attacking
water molecule adjacent to GTP at the active site.
GTP hydrolysis

29. The β subunit of the heterotrimeric G Protein has a
β-propeller structure, formed from multiple
repeats of a sequence called the WD-repeat.
The β-propeller provides a stable structural support
for residues that bind Gα.
It is a common structural motif for protein domains
involved in protein-protein interaction.
Gβ- side view of β-propeller
PDB 1GP2
Gβ – face view of β-propeller
PDB 1GP2

30. Two students or student groups should team up:
 Explore together the structure of an inhibitory Gα
with bound GTP analog GTPγS.
 Keep the display on one computer while together you
display Gαβγ-GDP on the other computer.
 Compare the position of switch II in the two cases.

31. The family of heterotrimeric G proteins includes
also:
 transducin, involved in sensing of light in the retina.
 G-proteins involved in odorant sensing in olfactory
neurons.
There is a larger family of small GTP-binding switch
proteins, related to Gα.

32. Small GTP-binding proteins include (roles
indicated):
 initiation & elongation factors (protein synthesis).
 Ras (growth factor signal cascades).
 Rab (vesicle targeting and fusion).
 ARF (forming vesicle coatomer coats).
 Ran (transport of proteins into & out of the nucleus).
 Rho (regulation of actin cytoskeleton)
All GTP-binding proteins differ in conformation
depending on whether GDP or GTP is present at
their nucleotide binding site.
Generally, GTP binding induces the active state.

33. A GAP may provide an essential active site residue,
while promoting the correct positioning of the
glutamine residue of the switch II domain.
Frequently a (+) charged arginine residue of a GAP
inserts into the active site and helps to stabilize the
transition state by interacting with (−) charged O
atoms of the terminal phosphate of GTP during
hydrolysis.
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase Activating
Proteins, promote GTP
hydrolysis.
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)

34.  Gα of a heterotrimeric G protein has innate
capability for GTP hydrolysis.
It has the essential arginine residue normally
provided by a GAP for small GTP-binding
proteins.
 However, RGS proteins, which are negative
regulators of G protein signaling, stimulate GTP
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)

35.  An activated receptor (GPCR) normally serves as GEF for a
heterotrimeric G-protein.
 Alternatively, AGS (Activator of G-protein Signaling)
proteins may activate some heterotrimeric G-proteins,
independent of a receptor.
Some AGS proteins have GEF activity.
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)
GEFs, Guanine Nucleotide
Exchange Factors, promote
GDP/GTP exchange.

36. Phosphatidylinositol Signal Cascades
Some hormones activate a signal cascade based on
the membrane lipid phosphatidylinositol.
O P
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OH
H
H
OHH
OH
H
O
H OH
1 6
5
43
2
phosphatidyl-
inositol

37. Kinases sequentially catalyze transfer of Pi
from ATP to
OH groups at positions 5 & 4 of the inositol ring, to
yield phosphatidylinositol-4,5-bisphosphate (PIP2
).
PIP2 is cleaved by the enzyme Phospholipase C.
O P
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OPO3
2−
H
H
OPO3
2−
H
OH
H
O
H OH
1 6
5
43
2
PIP2
phosphatidylinositol-
4,5-bisphosphate

38. When a particular GPCR (receptor) is activated, GTP
exchanges for GDP. Gqα-GTP activates Phospholipase C.
Ca++
, which is required for activity of Phospholipase C,
interacts with (−) charged residues & with Pi moieties of
the phosphorylated inositol at the active site.
O P
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OPO3
2−
H
H
OPO3
2−
H
OH
H
O
H OH
1 6
5
43
2
PIP2
phosphatidylinositol-
4,5-bisphosphate
cleavage by
Phospholipase C
Different isoforms
of Phospholipase C
have different
regulatory domains,
& thus respond to
different signals.
A G-protein, Gq
activates one form
of Phospholipase C.

39. Cleavage of PIP2
, catalyzed by Phospholipase C, yields
2 second messengers:
 inositol-1,4,5-trisphosphate (IP3
)
 diacylglycerol (DG).
Diacylglycerol, with Ca++
, activates Protein Kinase C,
which catalyzes phosphorylation of several cellular
proteins, altering their activity.
OHH2C
CH
H2C
OCR1
O O C
O
R2
diacylglycerol
OH
H
OPO3
2−
H
H
OPO3
2−
H
OH
H
H OH
OPO3
2−
1 6
5
43
2
IP3
inositol-1,4,5-trisphosphate

40. IP3
activates Ca++
-release channels in ER membranes.
Ca++
stored in the ER is released to the cytosol, where it
may bind calmodulin, or help activate Protein Kinase C.
Signal turn-off includes removal of Ca++
from the cytosol
via Ca++
-ATPase pumps, & degradation of IP3.
Ca++
ATP ADP + Pi
Ca++
IP3
calmodulin
endoplasmic
reticulum
Ca++
Ca++
-ATPase
Ca++
-release channel
View an
animation.

41. Sequential dephosphorylation of IP3 by enzyme-catalyzed
hydrolysis yields inositol, a substrate for synthesis of PI.
IP3 may instead be phosphorylated via specific kinases, to
IP4, IP5 or IP6. Some of these have signal roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells
stimulates Ca++
entry, perhaps by activating plasma
membrane Ca++
channels.
OH
H
OH
H
H
OHH
OH
H
H OH
OH
OH
H
OPO3
2−
H
H
OPO3
2−
H
OH
H
H OH
OPO3
2−
(3 steps)
+ 3 Pi
IP3 inositol

42. The kinases that convert PI (phosphatidylinositol) to
PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions 4
& 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of
phosphatidylinositol at the 3 position of the inositol ring.
O P
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OH
H
H
OHH
OPO3
2−
H
O
H OH
1 6
52
3 4
phosphatidyl-
inositol-
3-phosphate

43. Head-groups of these transiently formed lipids are ligands
for particular pleckstrin homology (PH) & FYVE protein
domains that bind proteins to membrane surfaces.
Other protein domains called MARKS are (+) charged, and
their binding to (−) charged head-groups of lipids like PIP2
is antagonized by Ca++
.
O P
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
OH
H
OH
H
H
OHH
OPO3
2−
H
O
H OH
1 6
52
3 4
phosphatidyl-
inositol-
3-phosphate
PI-3-P, PI-3,4-P2,
PI-3,4,5-P3, and
PI-4,5-P2 have
signaling roles.

44. Protein Kinase B (also called Akt) becomes activated
when it is recruited from the cytosol to the plasma
membrane surface by binding to products of PI-3
Kinase, e.g., PI-3,4,5-P3.
 Other kinases at the cytosolic surface of the plasma
membrane then catalyze phosphorylation of Protein Kinase
B, activating it.
 Activated Protein Kinase B catalyzes phosphorylation of
Ser or Thr residues of many proteins, with diverse effects on
metabolism, cell growth, and apoptosis.
 Downstream metabolic effects of Protein Kinase B include
stimulation of glycogen synthesis, stimulation of glycolysis,
and inhibition of gluconeogenesis.

45. Signal protein complexes:
Signal cascades are often mediated by large "solid
state" assemblies that may include receptors,
effectors, and regulatory proteins, linked together in
part by interactions with specialized scaffold proteins.
Scaffold proteins often interact also with membrane
constituents, elements of the cytoskeleton, and
adaptors mediating recruitment into clathrin-coated
vesicles.
They improve efficiency of signal transfer, facilitate
interactions among different signal pathways, and
control localization of signal proteins within a cell.

46. Lipid rafts:
 Complex sphingolipids tend to separate out from
glycerophospholipids & co-localize with cholesterol in
membrane microdomains called lipid rafts.
 Membrane fragments assumed to be lipid rafts are found to
be resistant to detergent solubilization, which has
facilitated their isolation & characterization.
 Differences in molecular shape may contribute to a
tendency for sphingolipids to separate out from
glycerophospholipids in membrane microdomains.
See diagram (in article by J. Santini & coworkers).

47. Signal complexes are often associated with lipid
raft domains of the plasma membrane.
Scaffold proteins as well as signal proteins may be
recruited from the cytosol to such membrane
domains in part by
 insertion of lipid anchors
 interaction of pleckstrin homology or other lipid-
binding domains with head-groups of transiently
formed phosphatidylinositol derivatives, such as PIP2 or
PI-3-P.

48. AKAPs (A-Kinase Anchoring Proteins) are scaffold
proteins with multiple domains that bind to
 regulatory subunits of Protein Kinase A
 phosphorylated derivatives of phosphatidylinositol
 various other signal proteins, such as:
 G-protein-coupled receptors (GPCRs)
 Other kinases such as Protein Kinase C
 Protein phosphatases
 Phosphodiesterases
AKAPs localize signal cascades within a cell.
They coordinate activation of protein kinases as well
as rapid turn-off of signals.