hanahan and weinberg (2000) cell 100: 57
DESCRIPTION
Signal Transduction Cross-Talk Plays a major Role in Biology. Hanahan and Weinberg (2000) Cell 100: 57. Sixteen (or more) Different Classes of Receptor Signaling Pathways. from Pollard & Earnshaw '02. Overview of MAP kinase pathways. Growth factors and caclium. Cytokines Cellular stress. - PowerPoint PPT PresentationTRANSCRIPT
1Hanahan and Weinberg (2000) Cell 100: 57.
Signal Transduction Cross-Talk Plays a major Role in Biology
2
Sixteen (or more) Different Classes of Receptor Signaling Pathways
from Pollard & Earnshaw '02
3
Overview of MAP kinase pathways
Growth factors and
caclium
Proliferation Differentiation Cell survival
Raf
MEK
ERK
Cytokines Cellular stress
Inflammation Cell death
MEKKs
MKKs
JNK/p38
Stimulus
Upstream Kinases
MAP Kinase
Response
Map KKK
Map KK
Map Kinase
4
Johnson & Lapadat Science 2003
Map Kinase phospho-relay systems
5
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.
6
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.
7
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
A bifunctional protein that inhibits K-Ras and also has protein phosphatase activity.
8
Ras GDP/GTP cycle
GTP
GDP
GAPPiGTPase
GAPS = RGS = Regulators of G-protein Signaling
9
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
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
cRAF1 = a tyrosine kinase
GAP = GTPase activating protein
MEKs = Map Kinase Kinases, unusual in that it will phosphorylate both
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)
GEF = Guanine Nucleotide Exchange Factor Raf
MEK1/2
ERK1/2
Rsk , MSK1,
etc
Mitogenesis, Differentiation,
Proliferation, development, neuronal survival,
Memory formation
Transcription Factors
Cytoskeletal proteins
SOS
RT
K
RT
K
Shc Grb2
Ras
Hormone
Classic “Map Kinase” Pathway
10
Nine classes of receptor tyrosine kinases
from Pollard & Earnshaw '02How does receptor occupancy yield activation?
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Dimerization/ Autophosphorylation Model
Schlessinger JBC 273:11987
First
Second
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Schematic of the PDGF receptor
Nature Reviews Molec Cell Biol 3; 177-186
Src
PI3K
P120RasGap
SHP2PLC-1
Q: Why autophosphorylation?
13
Diagram of SH2 domain
Site for binding P-tyr Site for binding AA side chain
Protein having SH2 binding site
Protein having specific P-tyr sequence
14
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)
from Waksman et al Cell 72:779
Specificity in the sequence surrounding the P-Tyr
15
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.
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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 Gs/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 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
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Graves et al. PNAS 90: 10300 Nov “93
Fig. 2 Forskolin inhibits PDGF-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:
MAPK (),
MAPKK (), and
PKA( ) activities.
Protein kinase A antagonizes PDGF-induced MAP kinase signaling in human arterial
smooth muscle
PKA
MAP kinase
MAP kinase kinase
0 -7 -6 -5 -4
100 -
80 -
60 -
40 -
20 -
0
% A
ctiv
ity
Forskolin
So! Which step in MapK pathway is
inhibited?
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PKA
TRENDS in Cell Biology Vol.12 No.6 June 2002
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, but might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4).
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 Src-dependent pathway (1). PKA might also inhibit Raf-1 by direct phosphorylation at serines 43, 259 and 621.
19
(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.
Graves and Krebs worked upstream from Raf-1 and 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.
Graves et al. PNAS 90: 10300 Nov “93
Raf-1 overcomes cAMP inhibition of MEK
MEK overcomes cAMP inhibition of MAP kinase
Q: how does this order the step for cAMP action?
20
Cook and McCormick worked downstream from receptor
cAMP DOES block Activation of Raf by Ras
D
CT blocks Erk-1 activity CT blocks proliferation
Grb-2/Shc binding
cAMP does NOT block activation of Ras by EGFR
Cook & McCormick Science 262: Nov ‘93
EGF auto-phosphorylation
cAMP does NOT block
21
Model of Rap1 activation by cAMP as negative regulator of ERKs
(a) Rap1 activation by cAMP inhibits ERKs. Hormonal stimulation of a Gs/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.
TRENDS in Cell Biology Vol.12 June 2002Q: how is Rap1 regulated by cAMP/PKA? Direct PO4?
So, this is likely one mechanism by which an inhibitory effect can occur!
Rap1 is a small GTP-binding protein having the same sequence at its effector domain as Ras. It therefore can inhibit Ras function
22
Probably not; Rap 1 Stimulation by cAMP Requires SRC Family
Kinases
Whoops, yet another player - src
23
PKA phosphorylates and activates src and active src
activates Rap1
From Stork et al, JBC Nov 02
Note use of S/D and S/A mutants and FLAG tagsWhat is purpose of S/D Src???
Iso/cAMP --> PO4 srcP- src will activate RAP1
Cbl = a src kinase
A B
24From Stork et al, JBC Nov 02
Mechanism for Src Stimulation of Rap1 Activity
25
cAMP can activate Rap1 to antagonize Ras signaling to Raf-1. cAMP activation of PKA activates Rap1 via an Src-dependent pathway (1). PKA might also inhibit Raf-1 by direct phosphorylation at serines 43, 259 and 621. PKA
TRENDS in Cell Biology Vol.12 No.6 June 2002
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, but might potentiate the activity of full-length Raf-1 through 14-3-3 binding (4).
Likely mechanisms of cAMP/PKA inhibition of ERK activation
26
Then what about B-Raf and cAMP Stimulation of MAPK?
PC-12 cells vs 3T3 cells
27
Vossler et al Cell 89:73 ‘97
cAMP-dependent activation of MAPK is potentiated by B-Raf
In 3T3 cells cAMP inhibits until B-Raf added
THM: Cell type specificity
In PC12 cells cAMP stimulates MAPK
28
What difference does it make whether or not a cell has
Rap-1/B-Raf?
Effect of EGF vs NGF on Duration of Action
29
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 Gs/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 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
30
Is this the whole story and what about activation of ERK by
cAMP dependent GEFs that occurs in some cells ?
31
Gefs: Direct binding of cAMP to Epac
deRooij & Bos Nature 396, 474 - 477 (1998)
cAMP binding Kd ~ 1 uM
Does binding do anything?
Active Epacs bind cAMP
32
From Kawasaki et al Science 282 Dec ‘98
Time course of Rap1A activation by cAMP-GEF (EPAC)
33
THM: There are many different mechanisms for regulation of Map Kinase pathways.Different cells and different parts of cell may utilize different mechanisms.
ER
K a
ctiv
ity
X? p90Rsk
Regulation of Raf/Map Kinases by PKA and GEFs
Xp90Rsk
cAMP
34
Ca2+
Ca2+
CRE
CREB CBP
pp
Transcription Neuroplasticity
p90rsk2 MSK1
MAPK
MAPK
B-Raf
Ras
MEK
Adenylyl Cyclase 1,8
CaM
cAMP
PKARap1
GEF
35
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-CREBPD 98059 is a MEK inhibitor. KCl depolarizes and increases intracellular free calcium. Impey et al (1998) Neuron 21: 869
36
Impey et al (1998) Neuron 21: 869
Calcium and cAMP Synergistically Stimulate of CREB-Mediated 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
37
Ca2+
Ca2+
CRE
CREB CBP
pp
Transcription Neuroplasticity
p90rsk2 MSK1
MAPK
MAPK
B-Raf
Ras
MEK
Adenylyl Cyclase 1,8
CaM
cAMP
PKARap1
GEF
38
PKA is Required for the Nuclear Translocation of Erk
Impey et al (1998) Neuron 21: 869
Rp is an Inhibitor of PKA
39
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.
40
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
41
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 remote memory