Tyrosine Kinases and Protein Tyrosine Phosphatases

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Tyrosine Kinases and Protein Tyrosine
Phosphatases
IND447 Signal Transduction Bob MooneyDept. of
Pathology and Laboratory Medicine
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Protein Tyrosine Kinase
Substrate ATP Substrate-P ADP
Protein Tyrosine Phosphatase (PTP)
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Tyrosine kinases - catalyze transfer of a
g-phosphate from ATP to the hydroxyl group on
tyrosine residues of protein substrates.
Tyrosine
Serine
Threonine
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Tyrosine Protein Phosphorylation

• Eukaryotic cells coordinate functions through
  environmental signals - soluble factors,
  extracellular matrix, neighboring cells.
• Membrane receptors receive these cues and
  transduce signals into the cell for appropriate
  response.
• Tyrosine kinase signalling is the major mechanism
  for receptor signal transduction.
• Tyrosine protein phosphorylation is rare (1)
  relative to serine/thrreonine phosphorylation.
• TK pathways mediate cell growth, differentiation,
  host defense, and metabolic regulation.
• Protein tyrosine phosphorylation is the net
  effect of protein tyrosine kinases (TKs) and
  protein tyrosine phosphatases (PTPs).

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Protein Tyrosine Kinases (TKs)

• Receptor tyrosine kinases (RTK)
• insulin receptor
• EGF receptor
• PDGF receptor
• TrkA
• Non-receptor tyrosine kinases (NRTK)
• c-Src
• Janus kinases (Jak)
• Csk (C-terminal src kinase)
• Focal adhesion kinase (FAK)

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Protein Tyrosine Phosphatases (PTPs)

• Receptor-like or Transmembrane PTPs
• CD45
• PTPa
• LAR
• Non-receptor or Cytoplasmic PTPs
• PTP1B
• SHP1
• SHP2

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Receptor Tyrosine Kinases
From Hubbard (2000) Annu. Rev. Biochem. 69,373.
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Non-Receptor (Cytoplasmic) Protein Tyrosine
Kinases
From Hunter (2001) Nature 411,355.
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Receptor Dimerization and Kinase Activation
From Hunter (2001) Nature 411,355.
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Activation Loop Conformations of the Insulin
Receptor
From Hubbard (2000) Annu. Rev. Biochem. 69,373.
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Stimulation of RTK Activity

• Activation loop is mobile but most conformations
  interfere with substrate binding.
• The juxtamembrane and C-terminal domains may
  exert additional restraints on activity.
• A subset of conformations is compatible with
  substrate and ATP binding and activity.
• Ligands initiate dimerization and
  transphosphorylation of the activation loop
  tyrosine residue(s).
• Autophosphorylation of the activation loop shifts
  its equilibrium to the active conformation,
  promoting substrate and ATP binding.
• Further autophosphorylation promotes binding of
  downstream signaling molecules.

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Insulin
glucose
Insulin Receptor
p110
p85
PI 3-kinase
Ras
IRS
SH3
Grb2
Akt
SH3
SHC
Glut 4
Insulin Receptor Substrate-1/2
Raf1
MAP kinase kinase
ERK1/2
mitogenesis
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p85
p110
From Hunter (2001) Nature 411,355.
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From Hunter (2001) Nature 411,355.
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Non-Receptor Tyrosine Kinases

• NRTKs associate with membrane receptors or
  multiprotein complexes which regulate their
  activity.
• Activation involves both conformational changes
  and tyrosine phosphorylation of activation loop
  residues by heterologous kinases or
  autophosphorylation.
• NRTKs contain domains that mediate binding to
  proteins, lipids, or DNA.
• proteins SH2, SH3, FERM
• lipids Pleckstrin homology (PH)
• DNA example, Abl

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Signalling Pathways Involving src Kinases

• Broadly expressed fyn, c-src, c-yes, and yrk
• hematopoietic lineages blk, c-fgr, hck, lck, and
  lyn.
• C-src associates with receptor and non-receptor
  tyrosine kinases via its SH2 domain
• PDGFR, EGFR, IGF-1R, FAK, CSF-1 etc.
• C-src associates with transmembrane proteins that
  are not kinases
• interleukin receptors, T and B cell receptors

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Structural Features of p60c-src
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C-Src Activation Mechanism
Extracellular signal
From Hunter (2001) Nature 411,355.
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Conformation of Inactive Src
Kinase CT lobe
From Hubbard (1998) J. Biol. Chem 273,11987
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Fatty Acylation of src Kinases

• Fatty acylation targets proteins to membranes
• Myristoylation - consensus N-terminal sequence
• Met-Gly-X-X-X-Ser/thr-
• Met is removed cotranslationally and myristate is
  linked via an amide bond
• necessary but not sufficient for membrane binding
  of all such proteins
• Two-Signal model for membrane binding of
  N-myristoylated proteins

Myristoylation palmitoylaiton
Myristoylation basis a.a.s
Myristoylation proteinprotein interaction
Myristoylation
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Fatty Acylation of src Kinases(cont)

• Palmitoylation - enzymatic transfer of palmitic
  acid (and other fatty acylCoAs) to cysteine
  residues
• Reactions occur at the membrane - mechanism of
  localization
• Increases binding of myristoylated src by 300
  fold
• Target sequence in src (and Ga)
• Met-Gly-Cys
• Mutation of Gly-2 or Cys-3 reduces membrane
  binding and plasma membrane targeting
• CH3(CH2)12COOH Myristic acid
• CH3(CH2)14COOH Palmitic acid

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IL-6 Signalling via the gp130/Jak/STAT Pathway
IL6
gp130
gp80
STAT 1
STAT 3
inactive
P
P
Regulation of gene transcription
STAT 1
STAT 3
Hetero- or homo-dimers