Transcript Signal Transduction Cross-Talk Plays a major Role in Biology Hanahan and Weinberg (2000) Cell 100: 57.

Signal Transduction Cross-Talk Plays a major Role in Biology
Hanahan and Weinberg (2000) Cell 100: 57.
1
Sixteen (or more) Different Classes
of Receptor Signaling Pathways
2
from Pollard & Earnshaw '02
Overview of MAP kinase pathways
Stimulus
Growth
factors and
caclium
Cytokines
Cellular stress
Raf
MEKKs
MEK
MKKs
MAP
Kinase
ERK
JNK/p38
Response
Proliferation
Differentiation
Cell survival
Inflammation
Cell death
Upstream
Kinases
Map KKK
Map KK
Map Kinase
3
Map Kinase phospho-relay systems
4
Johnson & Lapadat Science 2003
The JNKS are Stress-Activated Protein Kinases
1. JNKs bind to and phosphorylate the DNA binding
protein c-Jun and increase its transcriptional activity.
2. c-Jun is a component of the AP-1 transcription
complex, which is an important regulator of gene
expression.
3. AP-1 contributes to the control of many cytokine
genes and is activated in response to environmental
stress, radiation, and growth factors — all stimuli that
activate JNKs.
4. JNKs are important in controlling programmed cell
death or apoptosis.
5
The Small G proteins
1. The small G proteins are about half the size of Gs or Gi.
2. They are active when GTP is bound.
3. They have intrinsic GTPase activity that inactivates
them.
4. They serve as general coupling factors in a variety of
signaling systems and provide points for signal
transduction cross-talk.
6
G protein Modulators
1. GEFs: Guanylyl Nucleotide Exchange Factors
GEFs activate G proteins by stimulating the exchange of GTP for GDP. GEFs can
be activated by second messengers. For example there are calcium and cAMP
activated GEFs that stimulate Ras.
2. GAPs: GTPase Activating Proteins
GAPs stimulate the GTPase activity of G coupling proteins and inhibit G-protein
activity.
3. GDIs: GDP Dissociation Inhibitors
GDIs block dissociation of GDP from G proteins, thereby inhibiting G protein
activity.
4. SCOP: SCN Circadian oscillatory Protein
7
A bifunctional protein that inhibits K-Ras and also has protein phosphatase activity.
Ras GDP/GTP cycle
GTP
GDP
GAP
Pi
GTPase
GAPS = RGS =
Regulators of G-protein Signaling
8
Classic “Map Kinase” Pathway
Hormone
RTK = receptor tyrosine kinase
Shc
= adapter protein tyr P by growth factors contains PTB, SH2 domains
and tyr P sites
Grb2 = adapter protein with 1 SH2 & 2 SH3 domains
Shc
Grb2
SOS
SOS
= son of sevenless = GEF for Ras
Ras
= small G-protein, part of p21 Ras family including H-Ras, K-Ras,
N-Ras, & R-Ras
Ras
Raf
GAP
= GTPase activating protein
GEF
= Guanine Nucleotide Exchange Factor
cRAF1 = a tyrosine kinase
MEKs = Map Kinase Kinases, unusual in that it will phosphorylate both
MEK1/2
ERK1/2
Rsk , MSK1,
etc
thr and tyr (at least 7) MEK = MAPK/ERK Kinase
ERKs = Extracellular signal Regulated protein Kinase; MAP kinases
(mitogen activated kinases) phosphorylated on TEY motif (others
include JNKs, SAPKs, & p38 kinase)
Transcription Factors
Cytoskeletal proteins
Mitogenesis, Differentiation,
Proliferation, development, neuronal survival,
9
Memory formation
Nine classes of receptor
tyrosine kinases
How does receptor occupancy yield activation?
10
from Pollard & Earnshaw '02
Dimerization/
Autophosphorylation
Model
Schlessinger JBC 273:11987
First
Second
11
Schematic of
the PDGF
receptor
Q: Why
autophosphorylation?
Src
PI3K
P120
RasGap
Nature Reviews Molec Cell Biol 3; 177-186
SHP2
PLCg-1
12
Diagram of SH2 domain
Protein having
specific P-tyr
sequence
Site for binding P-tyr
Site for binding AA
side chain
Protein having
SH2 binding
site
13
Cutaway view of an SH2 domain bound to
the phosphopeptide, (PNY(P)EEI)
The space filling model
of the phosphopeptide
shows the
- backbone in yellow,
- side chains in green,
and
- phosphate in white.
Like a two pronged plug
the P-Tyr and isoleucine
fit into a two-pronged
socket on the surface of
the SH2 domain (red)
Specificity in the
sequence
surrounding the
P-Tyr
from Waksman et al
Cell 72:77914
Erk/MAP Kinase is Regulated by
cAMP
1. Cyclic AMP inhibits Erk/MAP kinase in most
non-neuronal cells and is generally
anti-proliferative.
2. Cyclic AMP stimulates Erk/MAP kinase in
neurons and plays a major role in
neuroplasticity.
This is an example illustrating that signal
transduction mechanisms are cell specific.
15
Model of Rap1 activation by cAMP as regulator of
ERKs in a cell type-specific manner
Fig. 2. (a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gas/cAMP/PKA module leads to Rap1 activation (GTP loading). Many cells
express Raf-1 as the major Raf isoform. In these cells, GTP-loaded Rap1 blocks Ras activation of Raf-1, thereby inhibiting growth factor activation of ERKs
and cell proliferation. (b) Some cells express B-Raf as well as Raf-1. In these cells, GTP-loaded Rap1 can activate B-Raf and the mitogen-activated
protein (MAP) kinase cascade and hormonal stimulation of cAMP/PKA/Rap1 in these cells activates ERKs. Rap1 might also antagonize Ras activation
of
16
Raf-1, as in (a). Rap1 activation of B-Raf often predominates over the inhibition of Raf-1, resulting in a net effect of ERK activation.
TRENDS in Cell Biology Vol.12 No.6 June 2002
Protein kinase A antagonizes PDGF-induced
MAP kinase signaling in human arterial
smooth muscle
Fig. 2 Forskolin inhibits PDGF100 -
% Activity
BB-induced activation of
MAPKK and MAPK and
activates PKA in a dose
dependent manner. Human
arterial SMCs were incubated
with forskolin or vehicle for 30
min. Cells then were stimulated
with 0. 3 nM PDGF for 5 min and
cell extracts assayed for:
PKA
80 -
60 -
MAPK ( ),
MAPKK (o), and
40 -
PKA( ) activities.
MAP kinase
20 MAP kinase kinase
0
0
-7
-6
-5
Forskolin
-4
So! Which step in
MapK pathway is
inhibited? 17
Graves et al. PNAS 90: 10300 Nov “93
Likely mechanisms of cAMP/PKA inhibition
of ERK activation
PKA
cAMP can activate Rap1 to antagonize Ras signaling to Raf-1. cAMP a
phosphorylation of serine 43 can inhibit the ability of Raf-1 to bind to GTP-loaded Ras (2). cAMP and PKA might
interfere with the activation of Raf-1 by activating the serine/threonine kinase Akt, which can also inhibit Raf-1 by
direct phosphorylation on serine 259 (3). PKA phosphorylation at serine 621 can inhibit isolated kinase domains,18
but
might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4).
TRENDS in Cell Biology Vol.12 No.6 June 2002
Graves and Krebs worked upstream from
Raf-1 and MEK
MEK overcomes cAMP
inhibition of MAP kinase
(A) Extracts of cells were incubated with purified
MAPKK, and MAPK activity was measured in
SDS/polyacrylamide gels containing MBP. The
intensities of the bands representing the MAPks,
(Erk1 and Erk2) were estimated by scanning the
exposed film with a densitometer. Activated
recombinant Erk2 was used as the standard.
Q: how does this order the step for cAMP action?
Raf-1 overcomes cAMP
inhibition of MEK
(B) Extracts from control, PDGF treated, control,
forskolin treated and PDGF/forskolin treated cells were
incubated with DE-53 ion exchange resin to prepare a
partially purified fraction of MAPKK. The DE-52
samples were incubated with activated Raf-1 and
Mg2+/ATP at 30o for 30 min, after which 5 ul was
removed for MAPKK assay. The rightmost represents
activated Raf-1 in the absence of cell extract.19
Graves et al. PNAS 90: 10300 Nov “93
Cook and McCormick worked downstream
from receptor
CT blocks Erk-1 activity
CT blocks proliferation
cAMP does NOT block
EGF autophosphorylation
cAMP does NOT block
activation of Ras by EGFR
cAMP DOES block
Activation of Raf by Ras
Grb-2/Shc
binding
D
20
Cook & McCormick Science 262: Nov ‘93
Model of Rap1
activation by
cAMP as negative
regulator of ERKs
Rap1 is a small GTP-binding protein
having the same sequence at its effector
domain as Ras. It therefore can inhibit
Ras function
(a) Rap1 activation by cAMP inhibits ERKs.
Hormonal stimulation of a Gas/cAMP/PKA
module leads to Rap1 activation (GTP
loading). Many cells express Raf-1 as the
major Raf isoform. In these cells, GTPloaded Rap1 blocks Ras activation of Raf1, thereby inhibiting growth factor
activation of ERKs and cell proliferation.
So, this is likely one mechanism
by which an inhibitory effect
can occur!
Q: how is Rap1 regulated by cAMP/PKA? Direct PO4?
21
TRENDS in Cell Biology Vol.12 June 2002
Probably not; Rap 1 Stimulation by
cAMP Requires SRC Family
Kinases
Whoops, yet another player - src
22
PKA phosphorylates and
activates src and active src
activates Rap1
A
Iso/cAMP --> PO4 src
B
P- src will activate RAP1
Cbl = a src kinase
Note use of S/D and S/A mutants and FLAG tags
What is purpose of S/D Src???
From Stork et al, JBC Nov 02
23
Mechanism for Src Stimulation of Rap1 Activity
From Stork et al, JBC Nov 02
24
Likely mechanisms of cAMP/PKA inhibition
of ERK activation
cAMP can activate Rap1 to antagonize
Ras signaling to Raf-1. cAMP activation
of PKA activates Rap1 via an Srcdependent pathway (1). PKA might also
inhibit Raf-1 by direct phosphorylation at
serines 43, 259 and 621. PKA
phosphorylation of serine 43 can inhibit the ability of Raf-1 to bind to GTP-loaded Ras (2). cAMP and PKA might
interfere with the activation of Raf-1 by activating the serine/threonine kinase Akt, which can also inhibit Raf-1 by
direct phosphorylation on serine 259 (3). PKA phosphorylation at serine 621 can inhibit isolated kinase domains,
25 but
might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4).
TRENDS in Cell Biology Vol.12 No.6 June 2002
Then what about B-Raf and
cAMP Stimulation of MAPK?
PC-12 cells vs 3T3 cells
26
cAMP-dependent activation of MAPK is
potentiated by B-Raf
In PC12
cells
cAMP
stimulates
MAPK
In 3T3
cells cAMP
inhibits
until B-Raf
added
THM: Cell type
specificity
27
Vossler et al Cell 89:73 ‘97
What difference does it make
whether or not a cell has
Rap-1/B-Raf?
Effect of EGF vs NGF on Duration of Action
28
Model of Rap1 activation by cAMP as regulator of
ERKs in a cell type-specific manner
Fig. 2. (a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gas/cAMP/PKA module leads to Rap1 activation (GTP loading). Many cells
express Raf-1 as the major Raf isoform. In these cells, GTP-loaded Rap1 blocks Ras activation of Raf-1, thereby inhibiting growth factor activation of ERKs
and cell proliferation. (b) Some cells express B-Raf as well as Raf-1. In these cells, GTP-loaded Rap1 can activate B-Raf and the mitogen-activated
protein (MAP) kinase cascade and hormonal stimulation of cAMP/PKA/Rap1 in these cells activates ERKs. Rap1 might also antagonize Ras activation
of
29
Raf-1, as in (a). Rap1 activation of B-Raf often predominates over the inhibition of Raf-1, resulting in a net effect of ERK activation.
TRENDS in Cell Biology Vol.12 No.6 June 2002
Is this the whole story and what
about activation of ERK by
cAMP dependent GEFs that
occurs in some cells ?
30
Gefs: Direct binding of cAMP to Epac
Active Epacs
bind cAMP
cAMP
binding Kd
~ 1 uM
Does binding do
anything?
31
deRooij & Bos Nature 396, 474 - 477 (1998)
Time course of Rap1A activation by
cAMP-GEF (EPAC)
32
From Kawasaki et al Science 282 Dec ‘98
Regulation
of Raf/Map
Kinases by
PKA and
GEFs
THM: There are
many different
mechanisms for
regulation of Map
Kinase pathways.
Different cells and
different parts of cell
may utilize different
mechanisms.
ERK activity
cAMP
X
?
X
p90Rsk
p90Rsk
33
Ca2+
Adenylyl
Cyclase 1,8
CaM
Ca2+
GEF
Ras
B-Raf
cAMP
Rap1
PKA
MEK
MAPK
MAPK
p90rsk2
MSK1
p
p
CREB
CRE
CBP
Transcription
Neuroplasticity
34
High Frequency Stimulation of CREB Phosphorylation in
Hippocampal Slices Is mediated Through Erk/MAPK
P-CREB was monitored using a phospho-peptide specific antibody that recognizes P-CREB
PD 98059 is a MEK inhibitor. KCl depolarizes and increases intracellular free calcium.
35
Impey et al (1998) Neuron 21: 869
Calcium and cAMP Synergistically Stimulate of CREBMediated Transcription in Neurons dep
KCl depolarization increases intracellular free calcium
Forskolin increases cAMP
CRE-mediated transcription was monitored using cultured neurons from a
CRE-lacZ reporter mouse strain
36
Impey et al (1998) Neuron 21: 869
Ca2+
Adenylyl
Cyclase 1,8
CaM
Ca2+
GEF
Ras
B-Raf
cAMP
Rap1
PKA
MEK
MAPK
MAPK
p90rsk2
MSK1
p
p
CREB
CRE
CBP
Transcription
Neuroplasticity
37
PKA is Required for the Nuclear Translocation of Erk
Rp is an Inhibitor of PKA
Impey et al (1998) Neuron 21: 869
38
Calcium Stimulates Erk/MAP Kinase in Neurons
1. Calcium activates adenylyl cyclases in neurons.
2. Calcium stimulates Ras through GEF’s.
3. Calcium stimulates the degradation of SCOP, a
negative regulator of Ras.
39
Mechanisms for Regulation of Adenylyl Cyclase
1. Calcium stimulation-mediated through calmodulin
Examples: AC1 and AC8
2. Calcium inhibition of type 3 adenylyl cyclase
Mediated through CaM Kinase II phosphorylation of AC3
3. Stimulation by Gs-coupled receptors
4. Inhibition by Gi-coupled receptors
5. Stimulation or inhibition by the beta/gamma complex of
G-coupling proteins
Examples: AC2 and AC4 are stimulated by beta/gamma.
6. Stimulation by protein kinase C
7. Inhibition by PKA
Example: AC5
40
Some Physiological Roles of the MAP Kinases
1. Ras/ Erk1,2 MAP Kinase and proliferation
2. Erk1,2 /MAP kinase and neuronal survival
3. Role of the Stress Activated JNK and p38 Kinases in
neuronal apoptosis
4. Role of Erk1,2 /MAPK in Circadian Rhythm in the
SCN
5. Cell cycle progression and Erk 5
6. Neuronal Cell Fate determination and Erk5
7. Memory Formation Erk1,2, Erk5 required for remote41
memory