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Lecture II
Protein kinases and cancers
December 18, 2008
Tyr kinases and phosphatases
Amino acid specificity of protein kinases:
(1) Ser/Thr-specific protein kinases
(2) Tyr-specific protein kinases
(3) His-specific protein kinases
(4) Asp/Glu-specific protein kinases
Classification of protein phosphatases:
(1) Ser/Thr-specific phosphatases
(2) Tyr-specific phosphatases
(3) Dual specificity phosphatases
(4) Acidic phosphatases
(5) Basic phosphatases
Conventional view of signal transduction
Protein Tyrosine Kinases: POSITIVE Regulators
Protein Tyrosine Phosphatases - NEGATIVE Regulators
S
Autophosphorylation
P
PTK
(Inactive)
PTP
PTK
(Inactive)
(Active)
S
P
PTP
P
P (Active)
Alternative view of signal transduction
Protein Tyrosine Phosphatases - POSITIVE Regulators
PTP
P
P
S
S
(Inactive)
(Active)
PTK
General concept –
Classification and basic function of
protein tyrosine kinases
Important references
1. Hunter (2000) Signaling-2000 and beyond. Cell, 100: 113-127
2. Schlessinger (2000) Cell signaling by receptor tyrosine kinases.
Cell, 103: 211-225
3.
Schlessinger (2002) Ligand-Induced, Receptor-Mediated Dimerization
and Activation of EGF Receptor, Cell, 110: 669-672
4.
Garrett et al., (2002) Crystal Structure of a Truncated Epidermal Growth
Factor Receptor Extracellular Domain Bound to
Transforming Growth Factor. Cell, 110: 763-773
Classification of human receptor tyrosine kinases (RPTKs)
Models for activation of
signaling proteins
A). By membrane translocation
B). By conformational change
C). By tyrosine phosphorylation
General concept for activation of receptor tyrosine kinases
through ligand-induced dimerization
Schematic depiction of the extracellular and
Transmembrane domains of HER1, a human EGFR
A loop located between
two EGF binding domains
Structure of the dimeric HER1 bound to transforming growth
factor alpha, which is a member of the EGF family
ligand
ligand
A loop located between
two EGF binding domains
Activation of receptor tyrosine kinases
Juxtamembrane
region
N-terminal
kinase lobe
C-terminal tail
Substrate precluding
loop
Substrate accessible
loop
Recruitment of intracellular signal-transduction proteins by binding to
pTyr residues in receptors or receptor-associated proteins
Classification of human cytoplasmic protine tyrosine kinases
Activation of c-Src
Two modes of intrinsic inhibition
by interactions between:
(1) SH2 domain and
phosphorylated Y527;
(2) SH3 domain and
polyproline region.
Surface model of a SH2 domain bound to a pTyr-containing peptide
Four-residue core sequence:
pTyr (0)-Glu1-Glu2-Ile3
Two pronged “plug”:
Side chain of pTyr and Ile
Homework:
Structural basis for a PTB domain
bound to a pTyr peptide
(Src PTK)
General concept for activation of cytokine receptors through
a cytosolic protein tyrosine kinase JAK
Activation of STAT (Signal Transduction and Activation of Transcription) protein
through phosphorylation and dimerization
?
SOCS:
suppressors of cytokine signaling,
induced by STAT proteins
Deregulation of pTyr signaling and
development of human cancers
*
*
*
*
*
*
Blume-Jensen and Hunter, Nature, 411: 355-365, 2001.
In most cases of chronic myeloid leukemia (CML), the leukemic cells share a chromosome
abnormality not found in any nonleukemic white blood cells, nor in any other cells of the
patient's body. This abnormality is a reciprocal translocation between one chromosome 9
and one chromosome 22. This translocation is designated t(9;22). It results in one
chromosome 9 longer than normal and one chromosome 22 shorter than normal. The latter
is called the Philadelphia chromosome and designated Ph1.
Expression of a
fusion PTK
p210 Brc-Abl
References:
* 1. Nat Cell Biol. 2001 Sep;3(9):785-92.
ErbB2, but not ErbB1, reinitiates proliferation and induces luminal
repopulation in epithelial acini.
Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge JS.
Department of Cell Biology, Harvard Medical School, 240 Longwood Ave,
Boston, MA 02115, USA.
2. Methods. 2003 Jul;30(3):256-68.
Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini
grown in three-dimensional basement membrane cultures.
Debnath J, Muthuswamy SK, Brugge JS.
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue,
Boston, MA 02115, USA. [email protected]
ErbB-2 is amplified and overexpressed
in 20-80% cases of early stage of
breast cancer.
Overexpression of ErbB-2 is correlated
with a poor clinical prognosis of
breast tumors.
Overexpression of ErbB-2 in cultured cells
induces both ligand-independent
receptor phosphorylation and cell
transformation.
The Her (ErbB) family of receptors and their ligands
To study the effect of ErbB-2 in morphogenesis
and oncogenesis of MCF-10A mammary
epithelial acini
-----Using MCF-10A cells grown in threedimensional basement membrane cultures as a
model system
A. A lobule from human mammary gland– a polarized architecture surrounding
a hollow lumen.
B. The morphology of MCF-10A acini cultured on basement membrane for 10 days.
The overlay method for 3D culture of MCF-10A cells on Matrigel
The Biological events during MCF-10A acinar morphogenesis
Expression of synthetic, ligand-inducible activation of ErbB receptors
(ErbB-1 or ErbB-2) in MCF10A cells
No AP1510
No AP1510
No AP1510
Activation of ErbB-1 by addition
Of AP1510
DAPI
Activation of ErbB-2 by addition of AP1510
DAPI
DAPI
Multi-acinar
Structure in
ErbB-2-activated
MCF-10A
DAPI
Re-initiation of proliferation in
growth-arrested, polarized
acini by activation of ErbB-2
ki-67 = proliferating-cell
antigen
Immunostaining with anti-Ki-67 antibody in MCF-10A acini
Activation of ErbB-1 does not disrupt epithelial cell polarity
HA: expression of ectopic ErbB receptors
ZO-1: tight-junction-associated protein
Gp135: Apical-membrane protein
Activation of ErbB-2 disrupts epithelial cell polarity
HA: expression of ectopic ErbB receptors
ZO-1: tight-junction-associated protein
Gp135: apical-membrane protein
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