Transcript ppt - Bio 5068

Growth Factors and Receptor Tyrosine Kinases
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RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
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 mg/kg
Mitogenic for fibroblasts
Regulates growth/differentiation of many target cells
Refs:
S. Cohen, JBC 237:1555, 1962
S. Cohen, Nobel lecture, 1986
Phospho-tyrosine signals
Kinases phosphorylate tyrosine (Y*) residues of target proteins
Y~P = 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)
A
X
TK
A
P
X
A
P
B
X
In its new proximity to A, B’s
activity (= X) can now:
Phosphorylate or de-phosphorylate
another protein
Make or degrade a 2nd messenger
Attract additional signaling molecules
B
Y~P provides long-lasting but erasable memory, which is
terminated by DE-phosphorylation
*Y = one-letter code for tyrosine; S = ser, T = thr, etc.
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
ALSO: TK-linked
receptors for:
Antigens (receptors on B and T cells
Growth hormone
Interleukin-4
Erythropoietin, many others
Alberts, 15-47
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. Y~P residues recruit other signaling proteins, generate multiple signals
EGF receptor as a model
1st RTK to be characterized
v-erbB oncogene = truncated EGFR
Evidence for EGFR dimerization
Yarden & Schlessinger
Rate of phosphorylation =
k[EGFR]2, 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)
How do we know that the EGFR autophosphorylates in trans?
Experiment: test WT and short EGFRs,
each with or without a kin- mutation
wt
kinshort kinshort kin+
+
+
+
+
+
+
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?
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
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.
T-loop
Cat. loop
Y1162 occupies the
active site
Substrate Y
sits in 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
Y1162
flips out
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
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
Signals generated
by the EGFR
Individual Y~P residues recruit
specific proteins, generate
different signals
The activated dimer phosphorylates itself
P .
SOS, a Ras GEF
.
P
T-loop only
P .
P
P
.
Multiple sites P
Docks via intermediate adapters to activate Ras
Ras activates multiple targets (MAPK)
PLC-g
Docking of Y-kinases allows Tyr-phos’n 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
P
P
P
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
Types of adapter interactions
A
B
P
C
Adapter 1
Y~P sequence motifs allow regulatable adapter functions
Also
SH2
PTB
Tyrosine phosphates
Tyrosine phosphates
SH3
Polyproline-containing sequences
PDZ
Pleckstrin homol. (PH)
Many others
Specific 4-residue sequences at C-termini
Phosphoinositides
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.
Lodish, 24-17
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)
How would you determine the
specificity of an individual SH2
domain for a phosphopeptide?
EGF activates the MAPK pathway in multiple steps,
with multiple mechanisms
EGF
Extracellular GF
EGFR
RTK
EGFR~P
Phospho-RTK
Grb2
Adapter
SOS
Ras
Small GTPase
Mechanism
Proximity
Allostery
Covalent modification
Ras-GEF
Raf
Ser kinase
Tyr/thr kinase
Mek
Ser kinase
Transcription factor
ERKs
C-Jun
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
EGFR Activation of Ras: Proximity & Allostery
The Players
RTK = EGFR
P .
P
P
.
P
P
Ras
GDP
P
“GF receptor binding 2”
Adapter, found in screen
for binders to EGFR~P
SH3
SH2
Grb2
SH3
SOS
“Rat Sarcoma”
Small GTPase,
attached to PM by
prenyl group
“Son of Sevenless”
GEF, converts Ras-GDP
to Ras-GTP
Found in Drosophila,
homol. To S.c. Cdc25
EGFR Activation of Ras: Proximity & Allostery
Even before EGF arrives . . .
.
.
Ras
GDP
SOS is “ready to go”:
already (mostly)
associated with Grb2 in
cytoplasm, in the resting
state
SH3
SH2
Grb2
SH3
SOS
EGFR Activation of Ras: Proximity & Allostery
Then . . . Covalent modification
P .
P
P
.
P
P
Ras
GDP
P
EGF-bound dimers
trigger phosphorylation,
in trans
SH3
SH2
Grb2
SH3
SOS
EGFR Activation of Ras: Proximity & Allostery
Then . . . Proximity
P .
P
P
.
P
P
P
SH2
Ras
GDP
SH3
Grb2
SOS
SH3
Grb2’s SH2 domain binds Y~P on EGFR,
bringing SOS to the plasma membrane
EGFR Activation of Ras: Proximity & Allostery
Then . . . Allostery
P .
P
P
.
P
P
P
SH2
Ras
GDP
SH3
Grb2
SOS
SH3
GDP
SOS now binds Ras-GDP, causing
GDP to dissociate, and . . .
EGFR Activation of Ras: Proximity & Allostery
Then . . . Allostery continues
P .
P
P
.
P
P
P
SH2
Ras
GTP
SH3
Grb2
SOS
SH3
GTP
GTP enters empty pocket on Ras, which
dissociates from SOS and converts
into its active conformation
EGFR Activation of Ras: Proximity & Allostery
Finally . . . Proximity again!
P .
P
P
.
P
P
P
SH2
Ras
GTP
SH3
Grb2
Raf
SOS
SH3
GTP
Ras-GTP brings Raf to the PM for
activation, and the MAPK cascade
is initiated
Raf
MAPK
Cascade
How does Ras activate Raf? Proximity vs. allostery?
Allostery: Ras recruits Raf to the PM and activates it directly
Ras
Ras
GTP
GTP
Raf*
Raf
MAPK Cascade
(Cytoplasmic)
Proximity: Ras recruits Raf to the PM, where it is activated by X
X
Ras
Ras
GTP
GTP
Raf
(Cytoplasmic)
Raf*
MAPK Cascade
How can we tell the difference?
Does Raf signal (without Ras) when recruited to the PM?
Stokoe et al. (1994) Science
Experiment
Attach a CAAX* box to Raf’s Cterminus
Express Raf-CAAx in cells,
measure activity of MEK,
an enzyme downstream
in the MAPK pathway
EXV
Raf
Raf+RasG12
RafCAAX
RafCAAX+Ras17N
RafCAAX+RasG12V
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)
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
Mammalian MAP Kinase Cascades
Johnson & Lapadat (2002) Science 298: 1911
Borrowed from Chan, STKE
The best understood MAPK cascade
MAPK = Mitogen-activated protein kinase
.
Raf-1
A-raf
B-raf
MAPKKK
Phos’n of T-loop
Ser residues
.
P
P
Phos’n of T-loop
Thr and Tyr
MEK1
MEK2
.
MAPKK
P
P
Phos’n of Ser/Thr
ERK1
ERK2
MAPK
C-Jun
Altered gene
expression
MAPK “cassettes” mediate many different responses
Vertebrates
Frog
oocyte
Mitogens
Progesterone
MAPKKK
MAPKKK
MAPKKK
MAPKK
MAPKK
MAPKK
MAPK
MAPK
MAPK
G2-M transition
Cell cycle arrest,
mating
Proliferation
S. cerevisiae
Mating pheromone
Additional sites for regulation
Different biology, similar
cassettes: why 3 kinases?
Combinatorial diversity
Magnitude amplification
Switch-like responses
Switch-like behavior*
Responses are not always graded
1.0
Progesterone
MAPKKK
Response
Instead . . .
Frog
oocyte
MAPKK
0.5
0
MAPK
0 1
5
Stimulus (multiples of EC50)
G2-M transition
Amplified sensitivity: reduces noise @ 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 22:288, 1997
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 halfmaximally 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.
Ferrell, et al., Science (1998)
Answer: All or nothing.
Of course, life is not so simple . . . BONUS slide
Does this work in
mammalian cells?
Blenis and co-workers
used FACS and
immunohistochemistry
(anti-DP ERK Ab) to look
at EGF activation of ERK
in Swiss 3T3 fibroblasts
MacKeigan MCB 2005
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.
HPK=hematopoeitic progenitor kinase DLK=dual lineage kinase (member of the MLK family)
Whitmarsh et. al. (1998) Science 281: 1671
Scaffolding roles of JNK-interacting proteins
Dhanasekaran (2007) Oncogene
Scaffold proteins involved in ERK-signaling
pathways
Dhanasekaran (2007) Oncogene
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases
EGFR Activation of PI3K combines Proximity & Allostery
P .
.
PIP2
P
SH2
P
P
Activated by
EGFR/p85
Can also be activated
by Rac or Ras!
PIP3
SH2
p85
p110
SH2
Recruitment from
cytoplasm to PM,
via SH2 domains
SH2
p85
p110
How do we know proximity is not enough?
1. p85 mutants that activate without binding to RTKs
2. Tethering to membrane does not activate
PI3-K pathway and Cancer Syndromes
RTK
GF
Cancer Syndromes
p
PIP3
PI3-K
PTEN
Akt1/2
(Tuberous Sclerosis
Complex)
(Target of rapamycin)
S6K
Lipid PTPase
Ser/Thr Kinase
Hamartin Tuberin
TSC2
TSC1
(Ras-homology
enriched in brain)
Lipid Kinase
Ras GAP
RheB
Small GTPase
mTOR
Kinase
4EBP-1
Protein synthesis
Cell growth/size/survival
Kinase
GI, Brain, Ovarian
Cowden’s, Multiple
Pancreas
TSC
Inhibitor of eIF4E
Kovich & Cohen (2004) Dematology Online Journal 10: 3.
Perelman (2004) Dematology Online Journal 10: 17.
PIP3 targets include many GEFs, many tyrosine kinases, and
others, including . . .
PKB (aka Akt) = ser/thr kinase that promotes cell survival
P
P
P
P
PIP3
PKB
(= membrane lipid)
PH
K
. . . is inactive in cytoplasm
. . . contains a PH (pleckstrin
homology) domain & a
kinase domain
Multi-step activation of PKB: proximity
PIP3
P
P
PH
P
K
PH domain recognizes 3’phosphate of PIP3, bringing
kinase domain to the PM
P
Proximity to PM
alone does not
activate the kinase
PH
K
Multi-step activation of PKB:
covalent modification
PIP3
P
P
PH
P
P
Inactive PKB
PDK1*
K
P
P
PH
P
P
K
P
P
Active (phos’d) PKB
*PDK1 is also recruited to the membrane via a PIP3-binding PH domain
Overall, two proximity steps plus (at least) one
phosphorylation step
EGFR Activation of PLCg combines THREE inputs
P .
.
P
P
P
PIP3
P
P
P
P
PIP2
P
P
PLCg (Inactive, in
cytoplasm)
PH
SH2
1. PROXIMITY:
Recruitment from
cytoplasm to PM,
via SH2 domains
SH2
Catalytic
EGFR Activation of PLCg combines THREE inputs
3. PROXIMITY:
Binds to PIP3 via
PH domain
P .
.
P
P
P
P
P
P
P
PIP2
DAG
P
SH2
P
2. COVALENT:
Activated by
EGFR phosph’n
PH
SH2
P
Catalytic
InsP3
Summary: Many RTK effectors require two
or more simultaneous inputs for activation
PI3K: recruitment via SH2, allosteric regulation by EGFR,p85
PKB: recruitment, phos’n by non-EGFR-kinase(s)
PLCg: recruitment, phos’n, retention at PM by binding PIP3
Why multiple inputs to each effector?
RTKs activate a complex network of interacting
response pathways (and this is the simple version!)
Active
P RTK P
P
P
STAT
PI3K
PLCg
DAG
InsP3
Rac
STAT~P
PKC
Targets
CaMK
PI3K
SOS
ROS
PTP
Ras
Cdc42
MAPK
JNK
Targets
Targets
Targets
Targets Targets
Targets
Targets
Targets
Targets Targets
Nuclear Transcription
Factors
PDK1
S6K
PKB
GSK3
Apoptosis
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
But how do you shut these things off?
Family of Protein Phosphatases
Tonks & Neel, Curr Op Cell Bio (2001)
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)
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)
Gleevec--proof that you can target kinases for
drug therapy
Goldman & Melo, NEJM, Oct 9, 2003
Gleevec--proof that you can target kinases for
drug therapy