Tyrosine Kinase Signaling

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Tyrosine Kinase Signaling I. Nonreceptor Enzymes
HANKS LECTURE II, 2003
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The human genome contains protein kinase 518
genes
Manning et al. (2002) Science 298, 1912-1934
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Tyrosine kinases (TK) account for 20 of the
human protein kinases
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Vertebrate Receptor Tyrosine Kinases
57 total in 20 families
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Vertebrate Nonreceptor Tyrosine Kinases
34 total in 10 families
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What is the normal cellular function of c-Src?

• Clues
• 1. Encoded by a proto-oncogene.
• - deregulated activity promotes aberrant
  proliferation

2. Expressed ubiquitously. - highest in
blood platelets, neurons, and osteoclasts -
suggests widespread function(s) not necessarily
related to proliferation

• 3. src -/- mice are surprisingly healthy, born
  and survive up to a year.
• - no obvious defects in platelet and
  neuronal function
• - major defect is in osteoclast function
  (bone resorption), leading to
• osteopetrosis (bone petrification)
• - compensation by other Src-family kinases?

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Clues to c-Src function, continued Expression
of Src family kinases Src Ubiquitous
neuron-specific isoforms Fyn Ubiquitous T
cell-specific isoform Yes Ubiquitous Lyn
Brain, B-cells, myeloid cells Hck Myeloid
cells Fgr Myeloid cells, B-cells Blk
B-cells Lck T-cells, NK cells, brain
4. src/fyn/yes triple knockout is embryonic
lethal at mid-gestation. - cells have
defects in motility but not proliferation -
motility defect rescued by reexpression of c-Src
5. c-Src and v-Src localize prominently to
cellular focal adhesions. - consistent with
role in cell motility
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Focal Adhesions
Specialized regions of the plasma membrane formed
at sites where cultured cells adhere tightly to
the underlying substratum
Focal Adhesion
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Tyrosine-phosphorylated proteins are enriched in
focal adhesions
Green actin Red pTyr Yellow costain (focal
adhesions)
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Integrins cell surface receptors for ECM ligands
Heterodimers of alpha and beta subunits. In
mammals, 18 alpha and 8 beta subunits have been
described which combine to form over 20 distinct
integrin heterodimers.
Large extracellular domains associate to form
the heterodimer and create ligand-binding site.
Divalent cations bound to one or both subunits
is essential for ligand binding.
Short cytoplasmic domains mediate linkages to the
actin cytoskeleton as well as to intracellular
signaling proteins.
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Some well-studied integrins ligands and
functions Integrin Major Ligand Cellular
Function(s) a5?1 Fibronectin Fibroblast
spreading and migration, cell
survival aIIb?3 Fibrinogen Platelet
aggregation, hemostasis av?3 Vitronectin Endothe
lial cell migration (angiogenesis) a6?4 Lami
nin Epithelial cell adhesion to basement
membrane
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Integrin-mediated cell-ECM adhesion promotes
tyrosine phosphorylation
1. Detach fibroblast cells by trypsin 2. Replate
on dishes coated with either fibronectin (pFN) or
poly-L-lysine (PLL, a negative control) 3.
After 30, lyse cells and assess phosphotyrosine
levels by immunoblotting with
anti-phosphotyrosine antibody
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What is it? A Src substrate?
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Identification of Src substrates
What are p210, p130, p125, etc?
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Parsons p125 Guans p120 Focal Adhesion
Kinase (FAK)
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Like FAK, CAS and paxillin are also focal
adhesion proteins and also emerged in screens
for Src substrates. They are typical docking
proteins, containing multiple protein-interaction
sites but no enzymatic activity.
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Crk crack
Crk CT10 regulator of kinase v-Crk
transformed cells exhibit increased tyrosine
phosphorylation of certain proteins e.g. CAS and
paxillin
Functions as an adaptor protein -- interactions
of SH2 and SH3 domains lead to recruitment of
signaling proteins to sites of tyrosine
phosphorylation
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Crk-interacting proteins
SH2
3
3
c-Crk
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FAK-Src-CAS-Crk/GEF Signaling Animation
Integrin clusters/ focal adhesions
Crk/GEF complexes recruited
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Downstream of Crk-associated GEFs
DOCK180
C3G
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What are the transformation-relevant substrates
of v-Src ?
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Cytokine signaling by the JAK-STAT pathway
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Cytokines Secreted proteins that stimulate cell
migration, growth, differentiation, and/or
survival of various blood cells including cells
of the immune system. - Examples Erythropoie
tin - regulates erythropoiesis (red blood cell
formation) - made AMGEN a ton of
Interferons - alpha, beta, and gamma
forms - functions include resistance to viral
infection Interleukins - over 20 distinct
forms - stimulate various cells of the immune
system including B and T cells,
megakaryocytes, neutrophils, macrophages
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Cytokine receptors
Consist of one or more single-membrane-spanning
molecules
IL6-R
INFg-R
EPO-R
gp gp 130 130
R1 R1
R2 R2
? a

• Signal through tyrosine phosphorylation, yet no
  tyrosine kinase domain in any subunit
• conserved membrane-proximal domain (shaded
  boxes) in one or more
• subunit is required for tyrosine phosphorylation
  and cellular responses
• - Membrane-proximal domains stably interact with
  Janus Kinases

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Janus Kinases (JAKs)
Named after Janus, the Roman God of Gates and
Doors. Janus has two facesone regarding what
is behind and the other looking toward what lies
ahead. Janus was also the God of Beginnings,
hence the first month January.
P
P
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Receptor
JAK/STAT pathway 1. Cytokine binding induces
receptor conformation change
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P
P
JAK/STAT pathway 2. JAKs activated by trans
autophosphorylation.
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JAK/STAT pathway 3. JAKs phosphorylate the
receptors and STATs are recruited. STAT
signal transducer and activator of transcription
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JAK/STAT pathway 4. JAKs phosphorylate the
STATs , causing them to dissociate and
dimerize through reciprocal SH2 interactions.
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JAK/STAT pathway 5. STAT dimers translocate to
the nucleus.
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Nucleus
JAK/STAT pathway 6. STAT dimers binds to specific
gene-regulatory elements and activate
transcription.
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Nonreceptor tyrosine kinases as receptor
subunits
Out
In
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END OF PART II