BIOM 200AB

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BIOM 200A/B Fall 2009 Module 4, Oct. 20 Protein
Kinases Lecturer Jean Y. J. Wang
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KEY POINTS

• Structure of the conserved protein kinase domain.
• Regulation of the protein kinase activity.
• Basic principles on protein phosphorylation-
• mediated signal transduction.
• 4. Organization of Fridays PBL sessions.

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Serine Threonine Tyrosine
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Tyrosine Kinases
The Protein Kinome
http//www.cellsignal.com/reference/kinase/index.j
sp
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3-D structure of a typical eukaryotic protein
kinase (ePK) catalytic domain two lobes (N
and C)  ATP-binding  catalytic loop
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GxGxxG
K
APE
DFG
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CDK2
PKA
ABL
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Upstream Activators
Kinase
Downstream Substrates
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How would you design a kinase for the purpose of
signal transduction?

• Build an inactive kinase that is activated when
  its structure
• is altered by a signal.
• b. Build an active kinase that is inhibited by a
  regulatory
• subunit, which dissociates in response to a
  signal.
• c. Build an inactive kinase that is activated
  upon
• phosphorylation by another kinase.
• d. Build an inactive kinase that is activated by
  a regulatory
• subunit, whose expression is increased in
  response to a signal.
• e. All of the above.

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How would you design a kinase for the purpose of
signal transduction?

• Build an inactive kinase that is activated when
  its structure
• is altered by a signal e.g., receptor tyrosine
  kinases (RTK).
• b. Build an active kinase that is inhibited by a
  regulatory
• subunit, which dissociates in response to a
  signal e.g.,PKA,
• SRC.
• c. Build an inactive kinase that is activated
  upon
• phosphorylation by another kinase e.g., ERK,
  JNK.
• d. Build an inactive kinase that is activated by
  a regulatory
• subunit, whose expression is increased in
  response to a signal
• e.g, Cyclin-dependent protein kinases, Cyclin
  E/Cdk2

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http//www.cellsignal.com/reference/kinase/index.j
sp
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Ligand-Induced Dimerization of RTK
Dimeric PDGF-AA, AB, or BB
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Current Model for EGF (monomeric)-Induced EGFR
Dimerization
asymmetric dimer of kinase domain
PMID 16777603 Zhang X. et al (2006)
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RTK Sub-Families
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How would you design a kinase for the purpose of
signal transduction?

• Build an inactive kinase that is activated when
  its structure
• is altered by a signal e.g., receptor tyrosine
  kinases (RTK).
• b. Build an active kinase that is inhibited by a
  regulatory
• subunit, which dissociates in response to a
  signal e.g.,PKA.
• c. Build an inactive kinase that is activated
  upon
• phosphorylation by another kinase e.g., ERK,
  JNK.
• d. Build an inactive kinase that is activated by
  a regulatory
• subunit, whose expression is increased in
  response to a signal
• e.g, Cyclin-dependent protein kinases, Cyclin
  E/Cdk2

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Auto-inhibitory regulation applies to a large
number of PKs in the kinome.
Inhibitory subunit the R-subunit of protein
kinase A tetramer
Auto-inhibition Catalytic activity inhibited by
intra-molecular interactions
pseudo-substrate auto-inhibitory assembly
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C-catalytic
R-Regulatory Binds cyclic AMP
Science 4 February 2005 Vol. 307. no. 5710, pp.
690 - 696 DOI 10.1126/science.1104607 Research
Articles Crystal Structure of a Complex Between
the Catalytic and Regulatory (RI) Subunits of
PKA Choel Kim,1 Nguyen-Huu Xuong,1,2 Susan S.
Taylor1,3,4
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active
inactive
Susan Taylor, Science 2005
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How would you design a kinase for the purpose of
signal transduction?

• Build an inactive kinase that is activated when
  its structure
• is altered by a signal e.g., receptor tyrosine
  kinases (RTK).
• b. Build an active kinase that is inhibited by a
  regulatory
• subunit, which dissociates in response to a
  signal e.g.,PKA.
• c. Build an inactive kinase that is activated
  upon
• phosphorylation by another kinase e.g., ERK,
  JNK.
• d. Build an inactive kinase that is activated by
  a regulatory
• subunit, whose expression is increased in
  response to a signal
• e.g, Cyclin-dependent protein kinases, Cyclin
  E/Cdk2

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• Protein Kinase Activators-
• Cyclins are activators of cyclin-dependent
  protein kinases
• (Cdks).
• 2. Small G-proteins, e.g., Ras-GTP, Rho-GTP,
  Rac-GTP
• are activators of Raf, ROCK, PAK kinases,
  respectively.
• 3. Protein kinases are activated by
  phosphorylation, e.g.,
• the MAP kinase cascade.

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KEY POINTS

• Structure of the conserved protein kinase domain.
• Regulation of the protein kinase activity.
• Basic principles on protein phosphorylation-
• mediated signal transduction.
• 4. Organization of Fridays PBL sessions.

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How can protein kinases influence their
substrates?

• Stimulate the enzymatic activities of their
  substrates.
• Inhibit the enzymatic activities of their
  substrates.
• c. Stimulate the interactions of their substrates
  with other
• proteins.
• d. Stimulate the poly-ubiquitination of their
  substrates for
• proteosome-mediated degradation.
• e. All of the above.

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Phosphorylation modifies the function of a
protein e.g., stimulating or inhibiting an
enzyme activity.
Phosphorylation creates a binding site for other
proteins.
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Phospho-epitope binding domains- SH2 binds
PTyr (tyrosine kinase signaling) PTB binds
PTyr (tyrosine kinase signaling)  14-3-3 binds
PSer/PThr (various pathways)  BRCT binds PSer
(DNA repair)  FHA binds PThr (various
pathways)  F-box binds PSer/PThr
(poly-ubiquitination)
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Examples of Proteins with SH2 Domain  Adaptors
GRB2, CRK,  Lipid modifying enzymes PLC-?,
PI-3 kinase (its regulatory subunit).  Intracel
lular tyrosine kinases SRC, ABL,..  Transcript
ion factors STATs.
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Auto-inhibitory assembly of Src tyrosine kinase
SH3 binds PXXP-motif
SH3
N-lobe
SH2 binds Y527P
SH2
C-lobe
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How to activate the auto-inhibited SRC tyrosine
kinase?
a. Proteolytic degradation of SH3 SH2
domains. b. Dephosphorylation of pY-527. c.
Dimerization. d. Phosphorylation of the
activation loop.
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Challenges-
Prediction of regulatory mechanisms for PKs
based on their primary amino acid sequences.
Kinome-wide analyses of cellular responses- How
many kinases are expressed, repressed,
activated, inactivated, by a given biological
perturbation?
Single cell assay for kinase functions Kinase
activity, kinase location and translocation,
substrate selection.
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KEY POINTS

• Structure of the conserved protein kinase domain.
• Regulation of the protein kinase activity.
• Basic principles on protein phosphorylation-
• mediated signal transduction.
• 4. Organization of Fridays PBL sessions.

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Module 4- Problem Based Learning Friday, Oct. 23,
2009
Four Teams, Four Topics, Two subgroups, Red
Blue, per Team
Team 1- 1, 5, 9, 13 17, 21, 25, 29, 33. Team 2-
2, 6, 10, 14 18, 22, 26, 30, 34. Team 3- 3, 7,
11, 15 19, 23, 27, 31, 35. Team 4- 4, 8, 12, 16
20, 24, 28, 32, 36.
Red groups will present to each other and Dr.
Newton. Blue groups will present to each other
and Dr. Wang. Each Team will prepare ONE
PowerPoint presentation, for a 20-min talk,
leaving 5-10 min for discussion.
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PBL Topics

• The ATM kinase in ionizing radiation-induced
  apoptosis.
• The VEGF receptor tyrosine kinase in tumor
  angiogenesis.
• The BCR-ABL tyrosine kinase in chronic
  myelogenous
• leukemia.
• 4. The LKB1 kinase and TSC1/2 in tumor
  suppression.