IGP Signaling 04: Tyrosine kinases I and II

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• IGP Signaling 04 Tyrosine kinases I and II
• Steve Hanks
• Cell and Developmental Biology
• Office U4206 MCN - Learned Labs
• Email steve.hanks_at_vanderbilt.edu
• Wednesday, Feb. 4th 900-1000am Src -- the
  prototypical tyrosine kinase
• 1000-1100am Functions of nonreceptor
  tyrosine kinases
• Reading
• Martin., G.S. (2001). The hunting of the Src.
  Nature Rev. Mol. Cell Biol. 2467-75.

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Src -- the protoypical tyrosine kinase
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Animal tumors can be caused by viruses - 1909
Long Island chicken farmer brings prize hen with
breast tumor (sarcoma) to Rockefeller Institute

• Peyton Rous induces sarcomas in other chickens
  by injecting them with cell-free and
  bacteria-free lysates from the original tumor

The causitive agent now known as "Rous
Sarcoma Virus" (RSV) Rous awarded Nobel Prize in
1966
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RSV induces neoplastic transformation Infected
cells acquire abnormal properties characteristic
of tumor cells
2 - Relaxed proliferation requirements a.
anchorage-independent proliferation (form
colonies in soft agar) b. loss of contact
inhibition (form foci in monolayer) c. reduced
growth factor requirement (proliferate in low
serum)
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Implication of cell transformation
Tumors can arise as a result of relatively simple
molecular changes within individual cells -- as
opposed to a complex process involving the
breakdown of whole tissues.
So how does RSV transform cells?
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RSV is a retrovirus (genetic material is RNA)
Adsorption to receptor
Penetration
Reverse transcription
Translation and assembly
Integration
Transcription
provirus
Budding
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RSV transformation is caused by a specific viral
gene called src
- 1970 Steve Martin isolates a
temperature-sensitive mutant of RSV
Martin
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The Enemy Within

• 1975
• Michael Bishop and Harold Varmus
• use nucleic acid hybridization to detect
• src-like genes in normal chicken cells

• - src probe prepared by subtractive hybridization
• of RSV to Vogts tdRSV
• other viral genes not detected in normal cells
• src genes were also found in mammals and a
• variety of other metazoans

Bishop
Varmus

• Conclusions
• The RSV src gene had a separate evolutionary
  origin from genes devoted to viral replication
• - Hypothesized that the src gene was (recently)
  transduced from the host cell genome!

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Key Insight - viral oncogenes are altered forms
of cellular genes (that may normally function in
control of cell proliferation) - later
confirmed by sequencing
Bishop and Varmus share Nobel Prize in 1989
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Questions
1. Why does v-Src protein transform cells and not
the endogenous c-Src?
- mutation or dosage?
- Ans. mutation, since c-Src overexpression is
not transforming
2. What is the normal function of c-Src protein?
3. What is the mutation in v-Src and how does it
alter protein function?
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v-Src is a protein kinase
Protein kinase assay of v-Src immunoprecipitates
(IPs)
Controls no kinase activity in IPs from
phenotypic revertant (shown), from normal cells
(not shown), or cells infected with RSV td
mutants (not shown)
Phosphoamino acid analysis indicated
phosphorylation was on threonine
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v-Src is a protein-tyrosine kinase
1980 Tony Hunter and Bart Sefton show that v-Src
(and c-Src) phosphorylates protein substrates
on tyrosine, NOT threonine.
I was too lazy to make up new buffer
Hunter
Sefton
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Phosphoamino acid analysis of 32P-IgG heavy chain
phosphorylated by v-Src (Hunter and Sefton)
- aging pH 1.9 buffer - pH drifts to 3.5 - pH
3.5 better separates pTyr from pThr during
electrophoresis - separation in 2-dimensions
helps separate pTyr from labelled nucleotides
(important for in vivo labelling)
pH 3.5
pH 1.9
Tyrosine phosphorylation of proteins is
discovered!
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Methodology Phosphoamino Acid Analysis (PAA)
1. Label phosphoprotein with 32P - in vitro
g-32PATP - in vivo 32P H3PO4
(orthophosphate)
2. Purify labelled phosphoprotein -
immunoprecipitate - SDS-PAGE/autoradiography -
elute from gel and TCA precipitate (or
immunoblot and excise radiolabelled band)
3. Limited acid hydrolysis to break peptide
bonds - dissolve protein in 6M HCl - incubate
110oC, 1 hr - remove HCl by evaporation
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HCl hydrolyzes peptide and phosphoester bonds,
releasing free amino acids and orthophosphate
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PAA continued
4. Separate products of hydrolysis by
electrophoresis on thin-layer cellulose plates
- dissolve acid hydrolysates in pH 1.9 buffer
containing large amounts of unlabelled pSer,
pThr, pTyr markers
- spot mixture on thin layer cellulose plate
- electrophorese in 1st dimension, pH 1.9
- turn plate 90o electrophorese in 2nd
dimension, pH 3.5
5. Visualize phosphopeptides by autoradiography
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Tyrosine phosphorylation is uncommon
Implication v-Src transforms by phosphorylating
specific cellular proteins on tyrosine
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Antibodies against phosphotyrosine
1981 Jean Wang and others develop antibodies
that recognize phosphotyrosine. Today
phosphotyrosine is almost always detected using
anti-phosphotyrosine antibodies.
Wang
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Anti- phosphotyrosine antibodies reveal putative
v-Src substrates
v-Src -
Figure shows anti-pTyr immunoblotting of normal
vs v-Src-transformed cells gt 50 distinct new
bands seen in RSV-transformed cells!
What are all of these v-Src substrate(s)? and
which one(s) cause transformation? Not easy
questions
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What is the mutation in v-Src and how does it
alter function? an easier question
early 1980s sequences of v- and c-src cDNAs are
compared
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1984 Several investigators show that
replacement of c-Src Ct-tail with v-Src Ct-tail
is sufficient to induce transformation Implicatio
ns
- c-Src Ct-tail appears to normally function by
suppressing kinase activity
- loss of the c-Src Ct-tail leads to deregulated
kinase activity and cell transformation
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Tyrosine phosphorylation regulates v-Src
and c-Src kinase activities
mid 1980s v-Src and c-Src are tyrosine kinase
substrates

• Tyr-416 is in the kinase domain,
• phosphorylation stimulates
• kinase activity

- Tyr-527 is missing in v-Src. Could this be a
critical regulatory site?
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1987 Mutation of Tyr-527 to Phe is sufficient to
activate both kinase and transforming activities
of c-Src
Conclusions - Tyr-527 phosphorylation
negatively regulates c-Src kinase activity -
v-Src transforms because it has lost Tyr-527 and
is thus constitutively-active
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Src homology domains
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SH1 is a eukaryotic protein kinase (ePK) domain
ePK domains transfer g-phosphate of ATP to
hydroxyl group of Tyr, Ser, or Thr residues on
protein substrate. Src SH1 only phosphorylates
Tyr.
I
II
III
IV
V
VIA
VIB
VII
VIII
IX
X
XI
N-terminal lobe (ATP binding)
C-terminal lobe (peptide binding and
phosphotransfer)
The ePK domain is encoded by 1-2 of all
eukaryotic genes
ePK domains with tyrosine-specific activity are
absent in yeast and plants, but account for
15-20 of the total in metazoans. Thus tyrosine
kinase activity appears to be a recent, but
successful, evolutionary development that
is implicated in signaling events associated with
metazoan complexity.
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SH2 (100 aa) and SH3 (60 aa) domains are
distinct modules found in a variety of signaling
proteins
Both function to mediate protein-protein
interactions
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SH2 domains bind to sites of tyrosine
phosphorylation
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SH2 domains bind tyrosine-phosphorylated proteins
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Results of pull-down - SH2 domains pull-down
pTyr-containing proteins - greatly enhanced by
v-Src expression
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1991 c-Src SH2 interacts with pTyr-527
- indicates possible mechanism for c-Src
autoinhibition
active
inactive
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1993-4 Zhou Songyang and Lewis Cantley show
specificity in pTyr site recognition by different
SH2 domains
Used recombinant SH2 domains (GST-fusions) to
purify phosphopeptides from a degenerate
phosphorylated peptide library
Cantley
Songyang
SH2 domain binding consensus Src pYEEI
Crk pYDXP Grb2 pYQNX
p85 (PI3K) pYMXM PLC-g (Ct) pYVIP
PLC-g (Nt) pYLEL underlined residues most
highly selected
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1993 Gabriel Waksman and colleagues report the
crystal structure of Src-SH2 in complex with the
pYEEI peptide
Waksman
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SH3 domains bind proline-rich peptide motifs
1993 SH3 domains are shown to bind Pro-X-X-Pro
motifs
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1994 Schreiber determines solution structures
(NMR) of Src-SH3 bound to class I and
class II peptides
Peptides adopt polyproline type II helical
conformation (3 residues/turn) Peptides bind to
shallow hydrophobic surface Binding stabilized
by - two hydrophobic pockets
accomodating PxxP prolines - acidic patch
(red) interacting with conserved Arg SH3
binding affinity is quite weak Kd in µM range
( 100-fold weaker than SH2)
class I peptide RALPPLP
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1997 Michael Eck determines crystal structure of
inactive c-Src
Key function of SH2-pTyr527 interaction is to
position SH3 domain
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Current Model for c-Src Activation
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Open questions
What is the normal function (substrates) of c-Src
protein?
How does v-Src (substrates) cause neoplastic
transformation?
Stay tuned