Growth Factors and Receptor Tyrosine Kinases

1
Growth Factors and Receptor Tyrosine Kinases

• RTKs--How do they work?
• EGFR signaling and ras
• MAP Kinase Cascades
• PI3K, PKB, PLCg
• PTPs (Protein Tyrosine Phosphatases)

2
Epidermal growth factor
Neurotrophic growth factor (NGF) isolated from
mouse submaxillary glands (Rita
Levi-Montalcini)
Stanley Cohen, 1962
Side effects of impure NGF preps
Premature eyelid opening (7d vs. 14 d)
Premature tooth eruption (6 d vs. 9 d)
Pure Tooth-lid factor EGF
Important roles in development
no EGF
EGF 1
g/kg
m
Mitogenic for fibroblasts
Regulates growth/differentiation of many target
cells
Refs
S. Cohen, JBC 2371555, 1962
S. Cohen, Nobel lecture, 1986
3
Phospho-tyrosine signals
Kinases phosphorylate tyrosine (Y) residues of
target proteins
YP target for distinctive protein binding
pockets, with surrounding sequences lending
specificity
ALWAYS activate by promoting proximity of
proteins A and B (sometimes by allostery also)
In its new proximity to A, Bs activity ( X)
can now
TK
X
X
X
Phosphorylate or de-phosphorylate another
protein
Make or degrade a 2nd messenger
Attract additional signaling molecules
B
YP provides long-lasting but erasable memory,
which is terminated by DE-phosphorylation
Y one-letter code for tyrosine S ser, T
thr, etc.
4
Phospho-tyrosine signals regulate growth
differentiation
RTKs Receptor Tyrosine Kinases
Extracellular region variable, with many
different motifs Usually cross membrane only
once Intracellular region contains conserved
catalytic domains
Alberts, 15-47
ALSO TK-linked receptors for
Antigens (receptors on B and T cells
Growth hormone
Interleukin-4
Erythropoietin, many others
5
How RTKs ( TK-linked Rs) work
1. Ligand promotes formation of RTK dimers, by
different mechanisms
Ligand itself is a dimer (PDGF)
One ligand binds both monomers (GH)
2. Dimerization allows trans-phosphorylation of
catalytic domains, which induces
activation of catalytic (Y-kinase) activity
3. Activated TK domains phosphorylate each other
and proteins nearby, sometimes on multiple
tyrosines
4. YP residues recruit other signaling proteins,
generate multiple signals
EGF receptor as a model
1st RTK to be characterized
v-erbB oncogene truncated EGFR
6
Evidence for EGFR dimerization
Yarden Schlessinger
Rate of phosphorylation kEGFR2, even in
micelles!
Therefore 2 EGFRs required for phosphorylation
Later confirmed by
Chemical cross-linking
FRET
Dominant-negative mutants (e.g., kinase-dead EGFR)
IMPORTANT
Dimerization/proximity alternative to allostery
(Shown by swapping EC/IC domains of EGFR, PDGFR)
7
How do we know that the EGFR auto-
phosphorylates in trans?
Experiment test WT and short EGFRs, each with
or without a kin- mutation
Honneger et al. (in vitro) PNAS 1989 (in
vivo) MCB 1999
Does this result rule out phosphorylation in cis
as well?
If not, how can you find out?
PS What do trans and cis mean?
8
How can we know that the EGFR does not
autophosphorylate in cis?
Need an EGFR that cannot homodimerize
EGFR family is huge, with many RTK members and
many EGF-like ligands
Such receptors often form obligatory heterodimers
with a similar but different partner
If A can dimerize only with A, then we can
inactivate the kinase domain of A and ask
whether A phosphorylates itself
Answer NO
QED
9
How does dimerization activate RTKs?
GFRs (like many kinases) have sites in their T
loops at which phosphorylation activates
Dimerization induces T-loop phosphorylation in
trans
Phosphorylation of Y (one or more) in T-loop
causes it to move out of the way of the active
site.
Proximity by itself is usually enough to promote
T-loop phosphorylation, but there may also be a
role for allostery
Once activated, each monomer can phosphorylate
nearby Y residues in the other, as well as in
other proteins
10
Bonus material
In contrast to most RTKs, phosphorylation of EGFR
activation loop is not critical to
activation. How does it work? Asymmetric dimers
activate by allosteric mechanism Zhang, et al
Cell, 2006
11
Growth Factors and Receptor Tyrosine Kinases

• RTKs--How do they work?
• EGFR signaling and ras
• MAP Kinase Cascades
• PI3K, PKB, PLCg
• PTPs (Protein Tyrosine Phosphatases)

12
Signals generated by the EGFR
The activated dimer phosphorylates itself
Individual YP residues recruit specific
proteins, generate different signals
T-loop only
Multiple sites
SOS, a Ras GEF
Docks via intermediate adapters to activate Ras
Ras activates multiple targets (MAPK)
PLC-g
Docking of Y-kinases allows Tyr-phosn of PLC-g,
which activates it
PI3-kinase
Adapters again
Docking allosterically activates PI3K
Each signal, in turn, activates a different set
of pathways, which cooperate to produce the
overall response
13
Adapters connect A with B, B with C . . . to
create complex, localized assemblies of
signaling proteins
Adapter 2
Each adapter has at least 2 interaction
domains, and may have other functions as well
C
B
A
Types of adapter interactions
Adapter 1
YP sequence motifs allow regulatable adapter
functions
SH2
Tyrosine phosphates
PTB
Tyrosine phosphates
Also
SH3
Polyproline-containing sequences
PDZ
Specific 4-residue sequences at C-termini
Pleckstrin homol. (PH)
Phosphoinositides
Many others
14
SH2 SH3 domains--src homology domains
SH domains are protein domains initially
discovered in Src, a transforming tyrosine kinase
found in Rous sarcoma virus. Sequences of many
signaling proteins that interact with RTKs
revealed multiple homologous domains to Src
region 2 and region 3. SH2 Protein motif of
100 amino acids, binds to phosphotyrosine
peptide sequences. (87 SH2 in the human
genome) SH3 60 amino acid domain, binds to
R-X-X-P-X-X-P peptide sequences. (143 SH3 in the
human genome)
Lodish, 24-17
How would you determine the specificity of an
individual SH2 domain for a phosphopeptide?
15
EGF activates the MAPK pathway in multiple steps,

with multiple mechanisms
EGF
Extracellular GF
EGFR
RTK
EGFRP
Phospho-RTK
Grb2
Adapter
SOS
Ras-GEF
Ras
Small GTPase
Raf
Ser kinase
Mechanism
Mek
Tyr/thr kinase
Proximity
ERKs
Ser kinase
Allostery
C-Jun
Transcription factor
Covalent modification
16
Fly genetics to the rescue
Fly eye consists of 800 ommatidia, an individual
lens structure consisting of 22 cells (8
photoreceptor cells, R1-R8) Eye development is
highly ordered process. RTK signaling is
essential. Mutation in sevenless results in loss
of R7. Additional mutations in pathway identified
sos (son-of-sevenless), boss (bride of
sevenless), Drk (downstream of receptor kinase)
Alberts, 15-53
17
EGFR Activation of Ras Proximity Allostery
The Players
RTK EGFR
.
P
Rat Sarcoma Small GTPase, attached to PM by
prenyl group
P
P
GF receptor binding 2 Adapter, found in screen
for binders to EGFRP
Son of Sevenless GEF, converts Ras-GDP to
Ras-GTP Found in Drosophila, homol. To S.c.
Cdc25
SH3
Grb2
SH2
SOS
SH3
18
EGFR Activation of Ras Proximity Allostery
Even before EGF arrives . . .
.
.
SOS is ready to go already (mostly)
associated with Grb2 in cytoplasm, in the
resting state
SH3
SOS
Grb2
SH2
SH3
19
EGFR Activation of Ras Proximity Allostery
Then . . . Covalent modification
EGF-bound dimers trigger phosphorylation,
in trans
SH3
SOS
Grb2
SH2
SH3
20
EGFR Activation of Ras Proximity Allostery
Then . . . Proximity
.
P
P
SOS
P
Grb2s SH2 domain binds YP on EGFR, bringing
SOS to the plasma membrane
21
EGFR Activation of Ras Proximity Allostery
Then . . . Allostery
.
P
P
P
GDP
SOS now binds Ras-GDP, causing GDP to
dissociate, and . . .
22
EGFR Activation of Ras Proximity Allostery
Then . . . Allostery continues
.
P
Ras
P
GTP
SOS
P
GTP
GTP enters empty pocket on Ras, which
dissociates from SOS and converts into its
active conformation
23
EGFR Activation of Ras Proximity Allostery
Finally . . . Proximity again!
.
Raf
P
Ras
P
GTP
SOS
P
GTP
Raf
Ras-GTP brings Raf to the PM for activation,
and the MAPK cascade is initiated
MAPK Cascade
24
How does Ras activate Raf? Proximity vs.
allostery?
Allostery Ras recruits Raf to the PM and
activates it directly
Raf
Raf
MAPK Cascade
(Cytoplasmic)
Proximity Ras recruits Raf to the PM, where it
is activated by X
X
Raf
Raf
MAPK Cascade
(Cytoplasmic)
25
How can we tell the difference?
Does Raf signal (without Ras) when recruited to
the PM?
Stokoe et al. (1994) Science
Experiment
EXV
Raf
Attach a CAAX box to Rafs C- terminus
RafRasG12
RafCAAX
RafCAAXRas17N
RafCAAXRasG12V
Express Raf-CAAx in cells, measure activity of
MEK, an enzyme downstream in the MAPK pathway
0
10
20
Relative MEK activity
CAAX (A aliphatic C cysteine) is a site
for prenylation prenylated proteins
concentrate at the PM
Answer proximity
Ras does localize Raf but does not activate it
(other proteins do)
26
Growth Factors and Receptor Tyrosine Kinases

• RTKs--How do they work?
• EGFR signaling and ras
• MAP Kinase Cascades
• PI3K, PKB, PLCg
• PTPs (Protein Tyrosine Phosphatases)

27
Mammalian MAP Kinase Cascades
Borrowed from Chan, STKE
Johnson Lapadat (2002) Science 298 1911
28
The best understood MAPK cascade
MAPK Mitogen-activated protein kinase
.
Phosn of T-loop Ser residues
Phosn of T-loop Thr and Tyr
MAPKKK
Phosn of Ser/Thr
MAPKK
C-Jun
MAPK
Altered gene expression
29
MAPK cassettes mediate many different responses
Frog oocyte
Vertebrates
S. cerevisiae
Mitogens
Progesterone
Mating pheromone
Cell cycle arrest, mating
Proliferation
G2-M transition
Additional sites for regulation
Different biology, similar cassettes why 3
kinases?
Combinatorial diversity
Magnitude amplification
Switch-like responses
30
Switch-like behavior
Responses are not always graded
Instead . . .
Amplified sensitivity reduces noise _at_ low
stimulus reversible
Bistable responses off or on, often via positive
feedback used for irreversible responses
(e.g., cell cycle)
Other examples?
JE Ferrell, Tr Bioch Sci 22288, 1997
31
All or nothing response in Xenopus oocytes
Progesterone, or fertilization, induces germinal
vesicle breakdown of Xenopus oocytes--a process
mediated by the MAPK cascade. Question At a
concentration of progesterone that half-maximally
activates MAPK (0.01 uM, panel A), are all the
oocytes activated halfway (panel B), or are half
of the oocytes activated fully (panel C)? Since
Xenopus oocytes are HUGE, one can look at MAPK on
a cell by cell basis. Answer All or nothing.
Ferrell, et al., Science (1998)
32
Of course, life is not so simple . . . BONUS slide
33
Scaffolds for MAP Kinase signaling
Deletion analysis of the binding of JIP-1 to
JNK1, MKK7, MLK3, and DLK. JIP-1 was expressed in
cells as a GST fusion protein together with HPK1
or epitope-tagged JNK1, MKK7, MLK3, and DLK (15,
16). The presence of these kinases in
glutathione-agarose precipitates was examined by
protein immunoblot analysis. HPKhematopoeitic
progenitor kinase DLKdual lineage kinase
(member of the MLK family)
Whitmarsh et. al. (1998) Science 281 1671
34
Elion (1998) Science 281 1625
35
Growth Factors and Receptor Tyrosine Kinases

• RTKs--How do they work?
• EGFR signaling and ras
• MAP Kinase Cascades
• PI3K, PKB, PLCg
• PTPs (Protein Tyrosine Phosphatases

36
EGFR Activation of PI3K combines Proximity
Allostery
PIP2
PIP3
.
.
P
P
P
P
Activated by EGFR/p85
SH2
Recruitment from cytoplasm to PM, via SH2
domains
p85
Can also be activated by Rac or Ras!
SH2
How do we know proximity is not enough?
1. p85 mutants that activate without binding to
RTKs
2. Tethering to membrane does not activate
37
PI3-K pathway and Cancer Syndromes
RTK
GF
Cancer Syndromes
PIP3
p
PI3-K
Lipid Kinase
GI, Brain, Ovarian
PTEN
Lipid PTPase
Cowdens, Multiple
Akt1/2
Ser/Thr Kinase
PANC
Tuberin
Hamartin
(Tuberous Sclerosis Complex)
TSC2
Ras GAP
TSC
TSC1
(Ras-homology enriched in brain)
RheB
Small GTPase
mTOR
(Target of rapamycin)
Kinase
S6K
4EBP-1
Inhibitor of eIF4E
Kinase
Protein synthesis
Kovich Cohen (2004) Dematology Online Journal
10 3.
Perelman (2004) Dematology Online Journal 10 17.
Cell growth/size/survival
38
PIP3 targets include many GEFs, many tyrosine
kinases, and others, including . . .
PKB (aka Akt) ser/thr kinase that promotes cell
survival
PIP3 ( membrane lipid)
PKB
. . . is inactive in cytoplasm
. . . contains a PH (pleckstrin
homology) domain a kinase domain
39
Multi-step activation of PKB proximity
PIP3
PH domain recognizes 3- phosphate of PIP3,
bringing kinase domain to the PM
Proximity to PM alone does not activate the
kinase
40
Multi-step activation of PKB covalent
modification
PDK1
PIP3
Inactive PKB
Active (phosd) PKB
PDK1 is also recruited to the membrane via a
PIP3-binding PH domain
Overall, two proximity steps plus (at least) one
phosphorylation step
41
EGFR Activation of PLCg combines THREE inputs
PIP2
.
.
P
PIP3
P
P
PLCg (Inactive, in cytoplasm)
P
P
P
SH2
1. PROXIMITY Recruitment from cytoplasm to
PM, via SH2 domains
SH2
42
EGFR Activation of PLCg combines THREE inputs
3. PROXIMITY Binds to PIP3 via PH domain
PIP2
DAG
.
.
PH
P
P
P
SH2
InsP3
P
P
P
SH2
Cata- lytic
2. COVALENT Activated by EGFR phosphn
43
Summary Many RTK effectors require two or more
simultaneous inputs for activation
PI3K recruitment via SH2, allosteric regulation
by EGFR,p85
PKB recruitment, phosn by non-EGFR-kinase(s)
PLCg recruitment, phosn, retention at PM by
binding PIP3
Why multiple inputs to each effector?
44
RTKs activate a complex network of interacting
response pathways (and this is the simple
version!)
Active RTK
P
P
P
P
STAT
PI3K
PI3K
PLCg
SOS
InsP3
DAG
Rac
PDK1
S6K
Ras
Cdc42
STATP
CaMK
PKC
ROS
PTP
MAPK
JNK
GSK3
PKB
Targets
Targets
Apoptosis
Targets
Targets
Targets
Targets
Targets
Targets
Targets
Targets
Targets
Nuclear Transcription Factors
45
Growth Factors and Receptor Tyrosine Kinases

• RTKs--How do they work?
• EGFR signaling and ras
• MAP Kinase Cascades
• PI3K, PKB, PLCg
• PTPs (Protein Tyrosine Phosphatases)

46
But how do you shut these things off? Family of
Protein Phosphatases
Tonks Neel, Curr Op Cell Bio (2001)
47
How Do PTPs dephosphorylate specific targets?
Intracellular targeting zip code model Extra
domains on PTPs confer localization and
protein-protein interactions Initially thought
that catalytic domains possessed little
specificity for RTKs. However, co-crystal
structures and biochemistry reveal that some PTPs
catalytic domains exhibit exquisite
sensitivity PTP-1B critical residues interact
with Insulin Receptor T-loop residues
Salmeen, et al Mol Cell (2000)
48
PTEN opposes PI3K by removing PI3-phosphate
PTEN discovered as a tumor suppressor
gene. Mutated in brain, breast and prostate
cancers. Has homology to dual specificity
phosphates, but shows little activity toward
phosphoproteins. Was discovered to remove
phosphates from PIPs thereby providing likely
mechanism for tumor suppression.
Cantley Neel, PNAS (1999)
49
Gleevec--proof that you can target kinases for
drug therapy
Goldman Melo, NEJM, Oct 9, 2003
50
Gleevec--proof that you can target kinases for
drug therapy
51
Bonus MaterialFly Eye- Cancer
MEN2 Genetic screen 3 fly transgenics
w.t., MEN2A MEN2B phenotypes
overproliferation, fate switches,
apoptosis, etc. flies can live without
eyes genetic modifier screen 93
enhancers, 47 suppressors Ras, JNK, Src,
etc.
RetMEN2B
Enhancer
Suppressor
52
Bonus Material Fly Eye- Cancer
MEN2 therapeutics new pathways
studying modifiers for role in overgrowth
(e.g. Src) drug screens growing flies
in 96-well plates screening small MW
compounds ZD6474 (Astra-Zeneca Sam
Wells)
wild type
RetMEN2B
RetMEN2B 0.2mM
RetMEN2B 1mM