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Many of the proteins have been discovered as Oncogene products and growth signaling proteins

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Title: Many of the proteins have been discovered as Oncogene products and growth signaling proteins


1
Tyrosine kinases and the Ras pathway
  • Many of the proteins have been discovered as
    Oncogene products and growth signaling proteins

2
Eukaryotic cell cycle
Increased cell mass
Cell division
3
The decision to divide is taken after a cell
samples its external environment, typically in
G1. For single cell organisms like yeast, this
decision is based on nutrient status. In
multicellular animals it is based on the
availability of Growth factors.
PDGF Platelet derived growth factor
S
G2
G1
G0
M
4
-increased cell mass occurs in G1 (Gap phase
1) -DNA duplication occurs in S phase -cell
division occurs in M (Mitosis) -G1 and G2 Gap
phases are required to coordinate duplication of
cellular components with cell division.
PDGF Platelet derived growth factor
S
G2
G1
G0
M
5
Question What happens in response to PDGF that
makes a G1 cell continue on in the cell cycle
towards S-phase?
Question What happens in response to PDGF that
makes a G1 cell continue on in the cell cycle
towards S-phase? Answer Signal Transduction!
6
Note that some hormones can cross the plasma
membrane and bind to cytoplasmic receptors. In
contrast, PDGF and many growth factors require a
cell surface receptor.
GF
Signal Transducer(s)
Responses (i.e. a new set of intracellular
signals)
7
Receptor Tyrosine Kinases
From Hubbard (2000) Annu. Rev. Biochem. 69,373.
8
Question What does PDGF do to cells?
Question What does PDGF do to
cells? Answer It depends on the cell type.
PDGF is released from platelets during wound
repair. It causes migration and proliferation
of fibroblast cells, and it causes the
recruitment of white blood cells.
9
Question How are these responses achieved?
Question How are these responses
achieved? Answer PDGF is a dimer, and
therefore, upon binding to PDGFR it induces
receptor dimerization. PDGFRs intracellular
portion contains a tyrosine (Y)-specific protein
kinase activity which is activated as a
consequence of receptor dimerization.
Activated PDGFRs autophosphorylate - creating
phosphotyrosine (pY) on themselves. These pY
sites are docking sites for signaling proteins to
bind.
10
Receptor Dimerization and Kinase Activation
From Hunter (2001) Nature 411,355.
11
SH2 (Src-homology domain 2) domains are protein
modules found in many signaling proteins. SH2
domains bind to pY sites on activated tyrosine
kinase receptors (like the PDGFR).
GF
KINASE
SH2-protein
-Y-P
P-Y-
P-Y-
PTB-protein
-Y-P
Responses
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Activated PDGFR binds to SH2 domains
in -non-receptor tyrosine kinases such as
p60Src (distinct substrates) -phospholipase Cg
which catalyses the breakdown of the lipid
phosphatidylinositol (4,5) bisphosphate
(PI4,5P2) into two second messengers, IP3which
causes release of Ca from the ER
intracellular store Diacylglycerol (DAG)
which activates protein kinase C
(PKC) -phosphatidylinositol 3 kinase.
Phosphorylates the lipid PI4,5P2 to generate the
second messenger PI3,4,5P3 -SHP2 tyrosine
phosphatase which dephosphorylates selected pY
residues on the receptor and other proteins, and
also connects to the Grb2Sos1 complex to
activate p21Ras -GTPase activating protein,
which inactivates p21Ras-GTP -Nck, which
regulated the cytoskeleton
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SH2 domain binding is sequence specific, i.e. the
specific residues surrounding each pY site
determine which signaling proteins bind to it
(thus, specific receptors can generate specific
signals)
EGF
PDGF
PTB binding
-570 pYIYV
Src (Fyn, Yes)
-581 pYVDP
pYLIP 992-
-740 pYMDM
p85 PI3K
Nck
-751 pYVPM
pYINQ 1068-
RasGAP
-771 pYMAP
NPVpYHNQ 1086-
Syp
NPDpYQQD 1148-
Grb2
-1009 pYTAV
pYLRV 1173-
PLCg
-1021 pYIIP
Shc
SH2 binding
17
O
Stearate
C
O
O
C
O
Arachidonate
O
O
P
O
O
P
O
O
O
5
6
O
OH
1
4
OH
OH
2
3
O
P
O
O
Phosphatidylinositol 4,5-bisphosphate or PIP2

(acyl chains are commonly but not exclusively
as drawn)
O
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p110
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PI3K A major form of PI3K is composed two
subunits the p85 regulatory subunit that
contains two SH2 domains and one SH3 domain, the
p110 catalytic subunit binds to p85. P110 is
very unstable without p85. P85, therefore
functions to stabilize p110 and also to inhibit
its catalytic activity in the absence of
pY-binding. After the PDGFR is activated, and
autophosphorylated, it binds p85. This
phosphotyrosine bound p85 activates the catalytic
activity of p110 to generate phosphatidylinositol3
,4,5triphosphate (PI 3,4,5 P3 or PIP3). PIP3
activates a number of PH domain containing
proteins to induce changes in the cytoskeleton,
cell cycle, and even cell survival. Note other
enzymes are capable of tightly regulating the
conversion of specific phosphatidylinositol
lipids from one type of second messenger to
another.
23
Phosphoinositide Target domains (in vivo)
PtdIns(3)P FYVE, PX (PH?) PtdIns(4)P PH
(OSBP/SAPP/CERT), EpsinR AP1 ?1 PtdIns(5)P none
identified PtdIns(3,4)P2 PHPtdIns(3,5)P2
none identifiedPtdIns(4,5)P2 PH, FERM, ANTH,
ENTH, tubby (PX?), AP2-, plus several
cytoskeletal proteins PtdIns(3,4,5)P3 PH
24
p110
25
From Hunter (2001) Nature 411,355.
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Ras GEFs Sos (Son of Sevenless) Tyrosine kinase
signaling RasGrp1 DAG signaling at the
Golgi RasGrf Ca signaling at the Plasma
membrane ER ( eg. NMDA Receptors) CN-RasGEF cAM
P responsive Ras GEF
29
Ras GEFs (guanine nucleotide exchange
factors) Under physiological conditions p21Ras
proteins bind both GDP and GTP very tightly and
spontaneous release of guanine nucleotides is
very slow (10-5 M/s). Sos proteins, which were
discovered in Drosophila, contain catalytic
sequences termed Cdc25 homology domains, which
possess specific guanine nucleotide exchange
activity for Ras. The Sos proteins also encode
several C-terminal PxxP motifs that bind to the
SH3 domain sequences in the small adapter
protein, Grb2 (SH3-SH2-SH3). This Grb2-Sos
interaction is required for linking tyrosine
kinase signals to p21Ras activation. The SH2
domain in Grb2 binds directly to specific
phosphotyrosine residues on activated growth
factor receptors, or to adapter proteins that
have been phosphorylated by activated tyrosine
kinases (for example the tyrosine phosphorylated
SHP2). Through such protein-protein interactions
the PH domain of Sos may become exposed and able
to interact with plasma membrane components as a
mechanism to facilitate Sos-Ras interaction.
Thus, many growth factors recruit a pre-existing
cytosolic Grb2-Sos complex to the plasma
membrane, where it can access p21Ras-GDP and
stimulate its conversion to p21Ras-GTP (i.e.
activation of Ras, or formation of Ras-GTP). Ras
activation occurs when Sos stimulates p21Ras-GDP
to release its GDP moiety. GTP then binds to the
nucleotide free p21Ras-Sos transition state
complex to create p21Ras-GTP plus free Sos. Note
other Ras exchange factors exist in addition to
Sos proteins.
30
Ras GAPs (GTPase activator proteins) P120
Ras-Gap p120 Ras-GAP, the first one identified
and cloned, binds and stimulates Ras' intrinsic
GTPase activity approx 105-fold returning Ras to
its OFF' state (p21Ras -GDP). p120 Ras-GAP has
two SH2 domains and binds directly to activated
PDGF receptorsmechanism to translocate to plasma
membrane. Until GAP functions, RasGTP accumulates
and interacts with 'effectors, proteins that
transduce the signal that began with the growth
factor receptor-Grb2-Sos interaction. Neurofibrom
in (NF-1) Type 1 neurofibromatosisPatients with
a loss of function mutation in the NF1 gene get
Neurofibromas and Schwannomas. These tumor cells
have an increased concentration of p21Ras-GTP
even though they still have p120 GAP protein. ?
complex regulation of Gap protein
function. CAPRI Ca(2)-dependent Gap which
inactivates the Ras-MAPK pathway
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Scaffold proteins mediate MAP kinase signaling
specificity
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Rho Family GTPases Ras is the prototype for a
super-family of small molecular weight G-proteins
that switch between structurally and functionally
distinct 'inactive' GDP-bound and 'active'
GTP-bound states. The Rho subfamily includes Rho,
Rac and Cdc42. They are activated downstream of
Ras and are important for the cellular responses
to growth factors. Like Ras, the Rho-family
GTPases are regulated by specific GEFs and GAPs.
Stimulation of cell cycle progression or
induction of cell transformation by Ras involves
several distinct downstream pathways one is the
well-established Raf-MEK-ERK kinase cascade
above another involves activation of the
Rho-family of GTPases. Activated GTP-bound forms
of these proteins are mitogenic and promote the
formation of focal adhesion complexes at the
plasma membrane and specific actin structures in
quiescent Swiss 3T3 fibroblast cells stress
fibres (RhoGTP), ruffles/ lamellipodia (RacGTP)
and filopodia/microspikes (Cdc42GTP).
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One of the mechanisms by which Rac is activated
in cells involves PIP3 stimulated activation of a
Rac-specific GEF protein. Surprisingly, the
Rac-GEF which stimulates the conversion of
Rac-GDP to Rac-GTP is Son-of-Sevenless (Sos has a
DBL/PH cassette). In this case the Sos is not
bound to Grb2 but instead it is bound to a
complex containing E3B1 and Eps8. When PDGF
activates its receptor, the Grb2-Sos complex
actives Ras. At about the same time, the SH2
domains of the p85 regulatory subunit of PI3K
binds to the PDGF receptor. This leads to
Ras-GTP phosphotyrosine synergistic activation
of p110 PI3K catalytic activity with subsequent
PIP3 formation. PIP3 then binds to the PH domain
of Sos and activates it as a Rac-specific GEF.
Once Rac-GTP is formed, then Rac effector
proteins are activated to induce membrane ruffle
formation (associated with rearrangement of the
actin cytoskeleton) as well as signaling through
Pak kinase and Map kinase-like pathways. If the
formation of the different complexes were to be
coordinated temporally or spatially, then a
convenient mechanism of regulation of signals
would ensue. Sos-1 would initially activate Ras
and subsequently be shifted to Rac, thus
determining the switching off of the upstream
signal, while allowing its propagation
downstream. Signaling from Ras to Rac and
beyond not just a matter of GEFs. Giorgio Scita,
Pierluigi Tenca, Emanuela Frittoli, Arianna
Tocchetti, Metello Innocenti, Giuseppina Giardina
and Pier Paolo Di Fiore. The EMBO Journal, Vol.
19, No. 11 pp. 2393-2398, 2000
48
Activating mutations in PI3K catalytic subunit
are common in cancer
49
PI3K activation is required for Ras-induced
cancer
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Peroxide Osmotic Shock
Inducer
Growth Factors
Cytokines/Cellular Stress
Cdc42
GTPase
Ras
Rac
PAK
MAPKKK
Mos, Raf
MEKK1, ASK, TAK, MLK
TAK
?
MAPKK
MEK1, 2
MKK4, 7
MEK5
MKK3, 6
MAPK
ERK1, 2
JNK1, 2 , 3
P38a, b, g, d
ERK5
Growth etc.
Apoptosis etc.
Inflammation etc.
?
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Negative Regulation of Receptor Tyrosine
kinase/P21Ras signaling
Question How is Receptor Tyrosine kinase/P21Ras
signaling negatively regulated ?
Question How is Receptor Tyrosine kinase/P21Ras
signaling negatively regulated ? Answer Just
about every way you could imagine! -tyrosine
dephosphorylation -ligand sequestration/receptor
antagonism -receptor internalization and
destruction -specific pathways can be blocked or
downregulated -inducible and constitutive
mechanisms
57
Tyrosine Phosphatases
Tyrosine Phosphatases All contain a conserved
Cx5R sequence required for catalysis.
Tyrosine Phosphatases All contain a conserved
Cx5R sequence required for catalysis. There
are four major classes of Tyrosine Phosphatases.
Tyrosine Phosphatases All contain a conserved
Cx5R sequence required for catalysis. There
are four major classes of Tyrosine Phosphatases.
1) The Tyrosine-specific Phosphatases 2) The
VH1-like Dual-specificity Phosphatases (includes
the Map kinase phosphatase that hydrolyzes
phosphotyrosine and phosphothreonine thus
inactivating Map kinase) 3) The Cdc25 family of
Tyrosine Phosphatases (dephosphorylate Tyrosine
15 and Threonine14 in cdc2 to activate it) and
4) The Low molecular weight Phosphatases.
58
Tyrosine Phosphatases All contain a conserved
Cx5R sequence required for catalysis. There
are two major classes of Tyrosine
Phosphatases That directly regulate RTK/p21Ras
signaling. 1) The Tyrosine-specific
Phosphatases 2) The Dual-specificity
Phosphatases, includes the Map kinase
phosphatase that inactivating Map kinase
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Receptor Tyrosine Phosphatases (RPTP)are active
as Monomers and inactivated through dimerization
(opposite of RTKs)
Active
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SH2 domain containing phosphatasese like
SHP2/corckscrew are inactive until they bind to
pY on PDGFR or other signaling proteins
SHP2/corckcrew can function to limit specific
signal transduction pathways downstream from
Receptor tyrosine kinases ironically this can
increase signaling
SHP2/corckcrew is also a substrate of PDGFR which
forms a binding site for Grb2Sos and thus helps
to activate p21Ras
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LAR A large Receptor-like Tyrosine Phosphatase.
LAR is a widely expressed receptor-like protein
tyrosine phosphatase that is implicated in
regulation of intracellular signaling triggered
by both cell adhesion and peptide growth factors.
Genetic studies revealed that LAR regulates
neuron axon path finding in Drosophila and
mammary gland epithelial cell differentiation in
mice. The molecular mechanism underlying the
tissue specific function of LAR has not been
clearly understood. We investigated the role and
mechanism of LAR in peptide growth factors EGF
and FGF signaling in human tissue culture cells
in which the expression of LAR is under the
control of an inducible promoter. We found that
although both EGF and FGF induce activation of
mitogen-activated protein kinase (MAPK), LAR only
inhibits FGF-induced MAPK activation. LAR does
not interact directly with the peptide growth
factor receptors, since the ligand-induced
autophosphorylation of growth factor receptors
was not affected by induction of LAR. The
specific effect of LAR on FGF-induced MAPK
activation appeared to be mediated by specific
inhibition of the phosphorylation of two signal
transducers that act downstream of the FGF
receptor, FRS2 and a 180 kDa protein, and by
prevention of their interaction with the adaptor
protein GRB2. In contrast, LAR selectively
inhibited the epidermal growth factor
(EGF)-induced phosphorylation of p130CAS and the
formation of the complex between p130CAS and GRB2
but this effect did not influence the activation
of MAPK by EGF. These data suggest that LAR and
similar receptor-like protein tyrosine
phosphatases may contribute to the regulation of
transmembrane signaling by selectively inhibiting
the tyrosine phosphorylation of specific signal
transducers that act downstream of the plasma
membrane-associated tyrosine kinases. The
consequent inhibition of the formation of
signaling complexes by these proteins may
contribute to the specificity of the signals
generated by specific peptide growth factors as
well as extracellular matrix proteins. Wang X,
Weng LP, Yu Q. Specific inhibition of FGF-induced
MAPK activation by the receptor-like protein
tyrosine phosphatase LAR. Oncogene 2000 May
419(19)2346-53
65
What happens afterwards? Down-regulation
of MAP kinase signaling
Mechanisms of down regulation and desensitization
of signaling
  • MAPK can phosphorylate upstream components of the
    pathway and attenuate their activity
  • Multiple phosphatases including dual specificity
    phosphatases (e.g. MKPs) counteract the activity
    of MAPK module at all levels. Often the synthesis
    of MAPK phosphatases is induced (STE-pathway,
    Drosophila JNK).
  • Secreted antagonists of the RTK can be induced
    and shut of the RTK in the a cell- non-autonomous
    manner.

66
Using powerful worm genetics, a mutation in the
Cbl gene (sli-1) was found to
suppress pathological effects of EGF Receptor
hypoactivation
Cbl is now known to Ubiquitinylate the
activated (autophosphorylated) EGFR and to send
it for destruction in the lysozome.
67
The Cbl E3 Ubiquitin ligase negatively regulates
Tyrosine kinase signaling
RF
Binds to specific p-Y residues
Ring Finger binds to E2 ubiquitin conjugating
enzymes
Binds to SH3 domains
Ubiquitin-associated domain
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Complex Feedback Regulation in Drosophila EGF-R
Signaling
Spitz
Negative Feedback
Positive Feedback
75
Extracellular matrix
FGF
-Y-P
-Y-P
-Y-P
P-Y-
-Y-P
GDP
GTP
-Y-P
P-Y-
-Y-P
-Y-P
FGFR
FRS2
Grb2
Sos1
p21Ras
Signal Transduction
Signal Transduction
76
Induction and tyrosine phosphorylation of
Sprouty Sprouty-Y-P competes Grb2Sos1 complex
off of FRS2 and promotes Gap inactivation of
p21Ras (not shown), thus blocking the Ras pathway
but not effecting other FGFR signaling
FGF
-Y-P
-Y-P
-Y-P
P-Y-
-Y-P
-Y-P
P-Y-
-Y-P
-Y-P
Sprouty
No p21Ras signaling
Signal Transduction
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Some major mechanisms by which Tyrosine
kinase/p21Ras Signaling is negatively
regulated Constituitive -Tyrosine phosphatases
(eg. SHP2 and LAR) -Destruction of Receptor
and/or signaling proteins (eg. Cbl E3
ligase) -Gaps (eg. p120RasGap and NF1) -Inositol
phosphatases (eg. PTEN) -Rho GTPase
Gaps -Serine/Threonine phosphatases -Proteins
involved in membrane trafficking of the
receptor -etc Inducible -receptor antagonists
(eg. Argos block ligand binding to the
EGFR) -Receptor suppressor (eg.
Kekkon) -Sprouty -Sef -MapK phosphatase
80
Additional Themes
81
Location, Location, Location
Ras signalling on the endoplasmic reticulum and
the golgi Chiu et al., Nature Cell Biology
4343-350 (2002). Current models evoke the
plasma membrane (PM) as the exclusive platform
from which Ras regulates signaling. We developed
a flourescent probe that reports where and when
Ras is activated in living cells. We show that
oncogenic H-Ras and N-Ras engage Raf-1 on the
golgi and that endogenous Ras and unpalmitoylated
H-Ras are activated in response to mitogens on
the golgi and endoplasmic reticulum (ER)
respectively. We also demonstrate that H-Ras that
is restricted to the ER can activate the Erk
pathway and transform fibroblasts, and that Ras
localized on different membrane compartments
differentially engages various signalling pathways
. Thus, Ras signalling is not limited to the PM,
but also proceeds on the endomembrane.
82
Phospholipase Cgamma activates Ras on the Golgi
apparatus by means of RasGRP1. Bivona TG, Perez
De Castro I, Ahearn IM, Grana TM, Chiu VK,
Lockyer PJ, Cullen PJ, Pellicer A, Cox AD,
Philips MR. Nature. 2003 424694-8 Ras proteins
regulate cellular growth and differentiation, and
are mutated in 30 of cancers. We have shown
recently that Ras is activated on and transmits
signals from the Golgi apparatus as well as the
plasma membrane but the mechanism of
compartmentalized signalling was not determined.
Here we show that, in response to Src-dependent
activation of phospholipase Cgamma1, the Ras
guanine nucleotide exchange factor RasGRP1
translocated to the Golgi where it activated Ras.
Whereas Ca(2) positively regulated Ras on the
Golgi apparatus through RasGRP1, the same second
messenger negatively regulated Ras on the plasma
membrane by means of the Ras GTPase-activating
protein CAPRI. Ras activation after T-cell
receptor stimulation in Jurkat cells, rich in
RasGRP1, was limited to the Golgi apparatus
through the action of CAPRI, demonstrating
unambiguously a physiological role for Ras on
Golgi. Activation of Ras on Golgi also induced
differentiation of PC12 cells, transformed
fibroblasts and mediated radioresistance. Thus,
activation of Ras on Golgi has important
biological consequences and proceeds through a
pathway distinct from the one that activates Ras
on the plasma membrane.
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Translation
85
Translation
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mRNA Stability
Feedback regulation of MAPK signaling by an
RNA-binding protein Sugiura R, Kita A, Shimizu
Y, Shuntoh H, Sio SO, Kuno T. Mitogen-activated
protein kinases (MAPKs) are evolutionarily
conserved enzymes that convert extracellular
signals into various outputs such as cell growth,
differentiation and cell death. MAPK phosphatases
selectively inactivate MAPKs by dephosphorylating
critical phosphothreonine and phosphotyrosine
residues. The transcriptional induction of MAPK
phosphatase expression by various stimuli,
including MAPK activation, has been well
documented as a negative-feedback mechanism
of MAPK signalling. Here we show that Rnc1, a
novel K-homology-type RNA-binding protein in
fission yeast, binds and stabilizes Pmp1
messenger RNA, the MAPK phosphatase for Pmk1
(refs 10, 11). Rnc1 therefore acts as a negative
regulator of Pmk1 signalling. Notably, Pmk1
phosphorylates Rnc1, causing enhancement of the
RNA-binding activity of Rnc1. Thus, Rnc1 is a
component of a new negative-feedback loop that
regulates the Pmk1 pathway through its binding to
Pmp1 mRNA. Our findings--the post-transcriptional
mRNA stabilization of a MAPK phosphatase mediated
by an RNA-binding protein--provide an additional
regulatory mechanism for fine-tuning of
MAPK signalling pathways. Nature 2003 424961-965
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Positive Feedback loop Activation of Sos by
Ras-GTP
Effect of RasGTP on the Nucleotide Exchange
Stimulation by SOScat(A) Comparison of intrinsic
(black) and SOScat calalyzed nucleotide exchange
reaction in the absence (orange) or presence of
stoichiometric amounts of RasY64A loaded with GTP
(red) or GDP (gray). RasmantGDP (1 µM) was
incubated in buffer containing 200 µ unlabeled
GDP in the absence or presence of SOScat (1 µM)
alone or in addition to nucleotide-loaded
RasY64A. Dissociation of mantGDP was monitored by
the decrease of fluorescence emission at 430 nm
over time.(B) Rates were fitted to single
exponentials. Error bars indicate standard
deviations of three independent experiments.
91
Positive Feedback loop Activation of Sos by
Ras-GTP
92
Cancer
Oncogenes -Ligands (eg. PDFR) -Tyrosine kinases
(receptors and non-receptor tyrosine
kinases) -Ras Gefs -Ras proteins -Raf
proteins -PI3K -Rho proteins -CblDN -???? Anti-On
cogenes -RasGaps (eg. NF1) -PTEN -?????
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