Biochem 503
November 25, 2008
Protein Tyr Kinases
Assigned reading (pdf are on-line)
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Tony Pawson (2004) Cell 116: 196-203
Joseph Schlessinger (2002) Cell 110: 660-672
Blume-Jensen & Hunter (2001) Nature 411:355-365
Protein Kinases in the Human Genome
Protein Tyr Kinases
Cell Signaling Technologies
<www.cellsignal.com>
Protein Ser/Thr Kinases
Protein Tyr Kinases - two classes
“Receptor”
i.e. transmembrane
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1. History of Protein Tyr Phosphorylation
read assigned Pawson article in Cell, 2004
A. Viral oncogenes; SRC and Cancer
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1917
Peyton Rous - avian sarcoma virus,
1960’s the src virus gene as causative agent
1976
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Peyton Rous
Varmus & Bishop show a c-src gene in humans
oncogene and proto-oncogene
- viral and endogenous
J. Michael Bishop and Harold Varmus
UCSF; Nobel Prize
the virus as an agent to distort
endogenous signaling that controls cell
proliferation
1978-1980
Ray Erikson (Univ. of Colorado) with postdoc Joan Brugge
(now both at Harvard)
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antisera from rabbits injected with RSV
IgG against the src protein: p60src
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Marc Collett and Erikson
src protein immunoppt. + ATP-32P, get 32P-IgG HC (55 kDa)
There’s a kinase activity in v-src!
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Tony Hunter (Salk Inst., LaJolla CA)
recognizes P-Tyr in ppt of PYmTAg using 2D TLE method
kinase activity, but unlike any known at the time.
HN
O
CH-CH2 P--O
O
O=C
O
NH
Phospho-Tyrosine
B. growth factor receptors
Stanley Cohen
Vanderbilt
(Nobel Prize)
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Ora Rosen
Einstein Med. Col
deceased 1990
Jos. Schlessinger
Tel Aviv, NYU, Yale
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Tony Pawson
Toronto
1980
EGF receptor, EGF found as hormone
action of EGF in A431 cells
found 32P-labeling of 150 kDa band
mistaken as P-Thr, then shown as P-Tyr
1982
insulin receptor kinase activity
Tyr auto-phosphorylation-multiple P-Tyr
sequence reveals pre-pro-protein,cleaved
and dimerized into a2b2
1983-85
technology of gas-phase peptide
micro sequencing
other GFRs shown to have PTK
activity: IGF-1, PDGF, FGF
v-erbB oncogene is cytoplasmic domain
of EGF receptor…..virus produces unregulated
version of growth regulator enzyme (PTK)
GRB = growth factor binding proteins……contain
SH2 domains that bind to P-Tyr
2. Analysis of Protein Phosphorylation
A. P-Tyr vs. P-Ser or P-Thr
2D method
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a. requires 32P-phosphoamino acid analysis, done by HCl hydrolysis
and HV-TLE in 2 dimensions, pH 1.9 & pH 3.5, now 1D at pH 2.5.
Non-quantitative due to different rates of hydrolysis
b. phospho-specific antibodies P-Tyr (4G10, PY20),
c. site-specific P-Tyr antibodies
(quantitation but no stoichiometry)
1D method
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B. Distribution of P-Tyr
a. species - metazoan enzymes…. only in multicellular organisms
P-Tyr in yeast, but no PTKs,
all P-Tyr from dual specificity kinases
b. subcellular -membranes >90%, in particulate cell fraction (B above),
mostly receptors + cytoskeleton or signaling complexes
c. P-Tyr proteins at UVA led to FAK (Parsons) and MAPK (Weber)
v-src targets in cells are in signaling pathways
3. Protein Tyr Kinases
A. Receptor Protein Tyr Kinases -(prototype EGFR)
1. Common Features
a. single pass transmembrane domain (alpha-helix)
b. single kinase domain - with or without 'insert’ (docking site)
c. variable and often large extracellular domain (hormone binding)
d. small but important juxta-(near) membrane segment (docking)
e. similar mechanism of activation (dimerization + phosphorylation)
2. Diversity of cell-surface proteins
a. many separate genes, wide repertoire (kinase and docking specificity)
b. complex extracellular domains, Ig and FNII domains
c. cell adhesion contacts-bidirectional signaling seen in Eph family
Receptor Protein Tyr Kinases - resembling EGFR
3. Activation Mechanism for Receptor PTKs
a. ligand-induced dimerization in membrane
b. Tyr phosphorylation of P-loop residues -->activity
c. phosphorylation of juxtamembrane and C terminal regions
d. recruitment of substrates and effectors
SH2 and PTB domains interact with P-Tyr
e. phosphorylation of recruited substrates: PI3K, PLCg IRS
f. internalization and other actions (nuclear)
Activation Mechanism for Receptor PTKs
Assignment: Explain how this model is not accurate,
relative to new structural data (see Schlessenger paper)
Mechanism for Receptor PTKs Downregulation
EGFR1 internalized, ErbB2 is not.
EGF induces degradation, TNFa gives recycling.
Inhibitors of Receptor PTKs in Clinical Use
Cetuximab
(Erbitux)
Gefitinib (Iressa)
Erlotinib (Tarceva)
Chemical Inhibitors of EGF Receptor Tyr Kinase
Currently in Use in Clinical Oncology - Oral Agents
Gefitinib = Iressa = ZD1839
Erlotinib = Tarceva = OSI774
Lapatinib = GW572016
Mutations in ATP binding site of EGF-R and ERB-B2
occur in tumors and produces resistance to inhibitors
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Int. J. Cancer (2005) 118: 257-262
B. Non-Receptor Protein Tyr Kinases (PTKs)
1. Common Features- prototype is the src family
a. kinase domain, (SH1) often near C terminus
b. targeting domains, most often SH2 [P-Tyr] and SH3 [Pro-XX-Pro]
c. auto-inhibited by multiple mechanisms (see below)
d. often lipid modified to localize at membrane;
palmitate, myristate
2. Diversity and Specificity
a. different tissue and developmental expression
b. different targeting because all SH3 and SH2 not equivalent
c. FERM domains send some PTKs to integrins
d. active site specificity revealed by selective inhibitors
Non-Receptor Protein Tyr Kinases (PTKs)
Development of Chronic Myeloid Leukemia
Chromosome
re-arrangement
produces an
activated
KINASE
BCL-ABL
to drive cell
proliferation.
Sussessful Targeted Therapy
Gleevec Cures CML Chronic Myeloid Leukemia
ATP
GleevecTM
BCR-ABL kinase
3. Activation Mechanisms for src-family PTKs
a. covalent - phosphorylation in P-loop; intrasteric effects
1. phosphorylation of P-loop to allow peptide substrate binding
AND
2. dephosphorylation of P-Tyr to relieve intrasteric P-Tyr::SH2
b. allosteric - constraints on C helix and ATP site
1. SH3 engagement to release conformational restraint
2. SH2 binding to compete away intrasteric interaction
Structure of src-family PTKs
Explain src activation using this diagram (next slide)
Activation Mechanisms for src-family PTKs
Orientation of the C Helix is Critical for Kinase Activity
positions the essential Glu91 for catalysis
C.
Dual-specificity Kinases [Tyr + Ser/Thr]
1. CDK-kinases that inactivate CDK (Myt1)
a. dual specificity for Tyr-Thr in CDK N terminal domain
b. regulators that prevent cell cycle progression
c. conserved in early eucaryotes…source of P-Tyr where no PTKs
2. MAP kinase kinases (MEK, for MAP/ERK kinase)
a. dual specificity for Tyr-X-Thr in activation loop, activate
substrate kinase
b. specificity for target kinase, MAPK, JNK, p38K, etc.
c. inhibited by non-competitive compounds…
..novel structure and action and specificity
Structure of Wee1 Kinase Complexed with Active Site Inhibitor
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Wee1 looks like a Ser/Thr kinase (in 1o and 3o structures)
but phosphorylates only Y15 in CDK2.
Structure of Dual-Specificity MEK1 Kinase with
MgATP plus Non-Competitive Inhibitor
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MEK1 Kinase: Non-competitive Inhibitor Produces
“Closed” Conformation with Displaced C Helix
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Comparison-Overlap of
PKA and MEK1
As a result of the change in
position of helix C, the side
chain of the highly conserved
glutamate residue Glu114
(equivalent to Glu91 in PKA)
is shifted away from the
active site and is unable to
form a critical ion pair with
the conserved catalytic Lys97
(equivalent to Lys72 in PKA).