Transcript Signaling9

Signal Transduction
serine (Ser)
threonine (Thr)
H
H
H3N+
C
COO
H3N+
C
COO
CH2
CH OH
OH
CH3
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).
O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase
 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.
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, and prevented from entering the nucleus.
O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase
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).
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.
NH2
cAMP
N
N
N
N
H2
5' C 4'
O
O
O
H
H 3'
O
P
O-
H
1'
2' H
OH
Phosphodiesterase enzymes
catalyze:
cAMP + H2O  AMP
N
N
The phosphodiesterase that
cleaves cAMP is activated by
phosphorylation catalyzed by
Protein Kinase A.
N
N
H2
5' C 4'
O
Thus cAMP stimulates its own
degradation, leading to rapid
turnoff of a cAMP signal.
NH2
cAMP
O
O
H
H 3'
P
O
O-
H
1'
2' H
OH
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
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.
R2C2 + 4 cAMP  R2cAMP4 + 2 C
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).
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 a-helices.
Rhodopsin was the first of these to
have its 7-helix structure confirmed
by X-ray crystallography.
Rhodopsin
PDB 1F88
 Rhodopsin is unique.
Lysozyme
insert
It senses light, via a bound
chromophore, retinal.
 Most 7-helix receptors have
domains facing the extracellular
side of the plasma membrane that
recognize and bind signal
molecules (ligands).
E.g., the b-adrenergic receptor
is activated by epinephrine and
norepinephrine.
ligand

b-Adrenergic
Receptor
PDB 2RH1
Crystallization of this receptor was aided by genetically
engineering insertion of the soluble enzyme lysozyme
into a cytosolic loop between transmembrane a-helices.
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.
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
 G-proteins are heterotrimeric, with 3 subunits a, b, g.
 A G-protein that activates cyclic-AMP formation
within a cell is called a stimulatory G-protein,
designated Gs with alpha subunit Gsa.
 Gs is activated, e.g., by receptors for the hormones
epinephrine and glucagon.
The b-adrenergic receptor is the GPCR for
epinephrine.
hormone
signal
outside
GPCR
The a subunit of
a G-protein (Ga)
binds GTP, and
can hydrolyze it
to GDP + Pi.
plasma
membrane
agga
AC
GDP bbGTP
GTP
GDP
cytosol
ATP cAMP + PPi
A and g 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.
hormone
signal
outside
GPCR
plasma
membrane
agga
AC
GDP bbGTP
GTP
GDP
cytosol
ATP cAMP + PPi
The sequence of events by which a hormone activates
cAMP signaling:
1. Initially Ga has bound GDP, and a,b, and g subunits
are complexed together.
Gb,g, the complex of b and g subunits, inhibits Ga.
hormone
signal
outside
GPCR
plasma
membrane
agga
AC
GDP bbGTP
GTP
GDP
cytosol
ATP cAMP + PPi
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 Ga becomes more accessible
to the cytosol, where [GTP] > [GDP].
Ga releases GDP and binds GTP (GDP-GTP exchange).
hormone
signal
outside
GPCR
plasma
membrane
agga
AC
GDP bbGTP
GTP
GDP
cytosol
ATP cAMP + PPi
3. Substitution of GTP for GDP causes another
conformational change in Ga.
Ga-GTP dissociates from the inhibitory bg complex and
can now bind to and activate Adenylate Cyclase.
hormone
signal
outside
GPCR
plasma
membrane
agga
AC
GDP bbGTP
GTP
GDP
cytosol
ATP cAMP + PPi
4. Adenylate Cyclase, activated by the stimulatory
Ga-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.
Turn off of the signal:
1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Ga causes it to rebind to
the inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterases catalyze hydrolysis of
cAMP  AMP.
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
b-arrestin.
• b-Arrestin promotes removal of the receptor from the
membrane by clathrin-mediated endocytosis.
• b-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.
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.
 Different isoforms of Ga have different signal roles. E.g.:
• The stimulatory Gsa, when it binds GTP, activates
adenylate cyclase.
• An inhibitory Gia, when it binds GTP, inhibits
adenylate cyclase.
Different effectors and their receptors induce Gia to
exchange GDP for GTP than those that activate Gsa.
 The complex of Gb,g that is released when Ga binds GTP
is itself an effector that binds to and activates or inhibits
several other proteins.
E.g., Gb,g inhibits one of several isoforms of Adenylate
Cyclase, contributing to rapid signal turnoff in cells that
express that enzyme.
 Cholera toxin catalyzes covalent modification of Gsa.
• ADP-ribose is transferred from NAD+ to an arginine
residue at the GTPase active site of Gsa.
• ADP-ribosylation prevents GTP hydrolysis by Gsa .
• The stimulatory G-protein is permanently activated.
 Pertussis toxin (whooping cough disease) catalyzes ADPribosylation at a cysteine residue of the inhibitory Gia,
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.
ADP
ribosylation
H
O
C
protein
NH2
O
+
N

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N
(CH2)3
NH
C
O
NH

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N

N
O P O CH2 N
O
O
H
H
H
H
+
NAD
OH
OH
(nicotinamide
adenine
dinucleotide)

O P O CH2
O
(CH2)3
H
NH
NH2
N
N
NH2+
+
N
H
N
O
O
C
N
H
H
OH
H
OH
H
protein
Arg
C
residue
NH2+
ADP-ribosylated
protein
NH2
nicotinamide
Structure of G proteins:
PDB 1GIA
The nucleotide binding site
in Ga consists of loops that
extend out from the edge of
a 6-stranded b-sheet.
Three switch domains have
been identified, that change
GTPgS
position when GTP
substitutes for GDP on Ga. Inhibitory Ga
These domains include residues adjacent to the terminal
phosphate of GTP and/or the Mg++ associated with the
two terminal phosphates.
O
GTP hydrolysis
N
NH
H
H
O
O

O
P
O
O
O
P
O
N
O
O
P
O
CH2
O
H
N
NH2
O
H
H
OH
H
OH
GTP hydrolysis occurs by nucleophilic attack of a
water molecule on the terminal phosphate of GTP.
Switch domain II of Ga includes a conserved
glutamine residue that helps to position the attacking
water molecule adjacent to GTP at the active site.
PDB 1GP2
PDB 1GP2
Gb - side view of b-propeller
Gb – face view of b-propeller
The b subunit of the heterotrimeric G Protein has a
b-propeller structure, formed from multiple repeats of a
sequence called the WD-repeat.
The b-propeller provides a stable structural support for
residues that bind Ga.
It is a common structural motif for protein domains
involved in protein-protein interaction.
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 Ga.
Small GTP-binding proteins include (roles indicated):
 initiation and elongation factors (protein
synthesis).
 Ras (growth factor signal cascades).
 Rab (vesicle targeting and fusion).
 ARF (forming vesicle coatomer coats).
 Ran (transport of proteins into and 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.
protein-GTP (active)
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase Activating
Proteins, promote GTP
hydrolysis.
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
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.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
 Ga 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
hydrolysis by Ga.
protein-GTP (active)
GDP
GEF
GEFs, Guanine Nucleotide
Exchange Factors, promote
GDP/GTP exchange.
GAP
GTP
Pi
protein-GDP (inactive)
 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.
Phosphatidylinositol Signal Cascades
O
O
R1
C
H2 C
O
O
C
CH
H2 C
R2
O
O
P
O
O
OH
2
phosphatidylinositol
H
H
1
6
H
OH
OH
H
OH
5
H
3
H
4
OH
Some hormones activate a signal cascade based on the
membrane lipid phosphatidylinositol.
O
O
R1
C
H2C
O
O
C
CH
H2C
R2
O
O
P
O
O
OH
2
H
PIP2
phosphatidylinositol4,5-bisphosphate
H
1
H
OH
3
H
6
OH
H
4
OPO32
5
H
OPO32
Kinases sequentially catalyze transfer of Pi from ATP to
OH groups at positions 5 and 4 of the inositol ring, to
yield phosphatidylinositol-4,5-bisphosphate (PIP2).
PIP2 is cleaved by the enzyme Phospholipase C.
O
Different isoforms
of Phospholipase C
have different
regulatory domains,
and thus respond to
different signals.
A G-protein, Gq
activates one form
of Phospholipase C.
O
R1
C
H2C
O
O
C
CH
H2C
cleavage by
Phospholipase C
R2
O
O
P
O
O
OH
2
H
PIP2
phosphatidylinositol4,5-bisphosphate
H
1
6
H
OH
OH
H
3
H
OPO32
5
H
4
OPO32
When a particular GPCR (receptor) is activated, GTP
exchanges for GDP. Gqa-GTP activates Phospholipase C.
Ca++, which is required for activity of Phospholipase C,
interacts with () charged residues and with Pi moieties of
the phosphorylated inositol at the active site.
OPO32 H
OH
2
H
1
6
H
OH
OH
H
3
H
OPO32
O
5
H
4
OPO32
IP3
inositol-1,4,5-trisphosphate
O
R1
C
H2C
O
O
C
R2
CH
H2C
OH
diacylglycerol
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.
Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
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, and degradation of IP3.
OPO32 H
OH
OPO32
OH
OH
H
H
OH
H
H
OPO32
H
IP3
(3 steps)
H
OH
OH
H
OH
OH
H
+ 3 Pi
H
H
H
OH
inositol
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.
O
O
R1
C
H2C
O
O
C
CH
H2C
R2
O
O
P
O
O
phosphatidylinositol3-phosphate
OH
2
H
H
1
6
OH
H
OPO32 H
3
H
4
OH
5
H
OH
The kinases that convert PI (phosphatidylinositol) to
PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions
4 and 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of
phosphatidylinositol at the 3 position of the inositol ring.
O
O
R1
C
H2C
O
C
CH
H2C
PI-3-P, PI-3,4-P2,
PI-3,4,5-P3, and
PI-4,5-P2 have
signaling roles.
O
R2
O
O
P
O
O
phosphatidylinositol3-phosphate
OH
2
H
H
1
6
OH
H
2
H
OPO3
3
H
4
OH
5
H
OH
Head-groups of these transiently formed lipids are ligands
for particular pleckstrin homology (PH) and 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++.
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.
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.
Lipid rafts:
 Complex sphingolipids tend to separate out from
glycerophospholipids and 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 and
characterization.
 Differences in molecular shape may contribute to a
tendency for sphingolipids to separate out from
glycerophospholipids in membrane microdomains.
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 lipidbinding domains with head-groups of transiently
formed phosphatidylinositol derivatives, such as
PIP2 or PI-3-P.
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.