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Nadia Dubé , Alan Cheng * and Michel L. Tremblay McGill Cancer Centre and Department of Biochemistry, McGill University, 3655 Promenade Sir-William-Osler, Room 715, Montreal, QC, Canada H3G 1Y6
The role of protein tyrosine phosphatase 1B in Ras signaling
Dubeet al. (2004) Proc. Natl. Acad. Sci. USA 101, 1834-1839
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Protein Tyrosine phosphatases (PTPs)
• ~30 PTPs in human genome
• Remove phosphorylation from tyrosine residues
Recall
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Protein tyrosine phosphatase 1B (PTP1B)
• Removes phosphorylation from tyrosine residues
•Negative regulator or several RTKs
•A regulator of metabolism
•Reduces insulin, leptin and growth hormone signaling
•>30 protein tyrosine kinases linked to malignant transformation and cancer
•Overexpression and/or mutations
Thus, if down-regulate dephosphorylation events- predict increased cancer
The question- If inhibit dephosphorylation, will cancer increase??
Background
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PTP1B
Recall Receptor tyrosine kinases (RTKs)
Background
•If inhibit PTP1B - Will cancer increase??
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ERK
1- Is receptor tyrosine phosphorylation increased?
3- Is Ras activated?
2- Is cell proliferation increased?
4. Is p120RasGAP activated?
5. Is p120RasGAP mRNA increased as well?
In PTPB1-/ - mice….
6. Is p120RasGAP 1 a substrate of PTP1B??
AKT
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•PTPB1 deficient mice (PTPB1-/ -) are resistance to diabetes and obesity
Evidence
• IGF-1, EGF and PDGF receptor levels increase in PTPB1-/- mice
But… no increased tumorigenesis observed
Why the discrepancy??
•Cell-specific differences in RTKs?•Tumorigenesis progression, but not initiation increased?•PTP1B activates oncogenesis downstream of RTKs
…But will PTPB1-/ - mice have increased tumor production?
Possibilities
Background
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Fig. 1. Differential regulation of PDGF-
signaling pathways by PTP1B
cellular tyrosine phosphorylation increased
PDGF stimulation:
AKT phosphorylation increased (more in PTP1B-/- cells)
Erk phosphorylation decreased in PTP1B knockout cells
Conclude- PTP1B seems be selective
Is tyrosine receptor phosphorylation increased in PTPB1-/ - mice?
Thus, some proteims are (AKT), some aren’t (ERK)
WT KO
ERK AKT
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Primary Ab (Rabbit Anti-1-antitrypsin)
A1-antitrypsin
Secondary Ab(Anti-rabbit Ab)
Alkaline peroxidase
Components of a Western analysis
Polyacrylamide gel elecrophoresis, transfer to nylon
membrane
1. Protein separation
2. Detection
1-AT protein
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Fig. 2. Decreased cell growth and Ras activity of SV40 TAg transformed fibroblasts lacking PTP1B
decreased monolayer growth in PTP1B-/- cells
10% FBS1% FBS
2- Is cell proliferation increased?
Answer- No, actually decreased.
KO KOWT WT
Cel
l pro
lifer
atio
n
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Fig. 2. Decreased cell growth and Ras activity of SV40 TAg transformed fibroblasts lacking PTP1B
PDGFR expression is increased in PTP1B-/- cells
diminished Ras-GTP levels In PTP1B-/- cells
3- Is Ras activated in PTPB1-/ - mice?
Answer- No, actually decreased activity.
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Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression
Answer- Expression levels of the adapter proteins Shc or Grb2, or the kinases (Src and Erk) (data not shown)
What is not changed in PTPB1-/- cells?
p120RasGAP elevated…
…but p190RhoGAP unchanged
p120RasGAP decreased when re-introduce PTP1B
What is changed?
…but no effect on another knockout cell line
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•Elevated in PTPB1-/- cells
•Normal levels when introduce in PTPB1 gene into cells
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Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression
p120RasGAP mRNA is
elevated in two
independent cell lines
Is p120RasGAP mRNA increased as well?
Carry out RT-PCR reaction…
SV40-immortalizedSpontaneously -immortalized
…then load product onto agarose gel electrophoresis
Conclude: PTPB1 suppresses RasGAP gene expression
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Fig. 4. p62Dok is a putative substrate of PTP1B
Question- Is p120RasGAP 1 a substrate of PTP1B??
GST-PTPB1Glutothione
Wash away unbound
Load bound onto gel
Use antibodies to detect what binds PTPB1
Conclude- No, but p62Dok is.
GST Pulldown experiment
anti-pY
anti-p62Dok
D181A mutant
GST-tagged PTP1B (WT or D181A)
NIH3T3 cell
Cell lysate
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Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras
Growth in Soft agar
Overcome Contact inhibition
If introduce dominant active Ras mutant (V12Ras)….
….can transform fibroblasts, even in
the absence of PTP1B.
Tw
o p
rop
ert
ies
of
tra
nsf
orm
ed
ce
lls
Will Ras activation alone really cause cell proliferation?
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Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras
Growth in Soft agar
Overcome Contact inhibition
Mock
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Fig. 6. Model of impaired Ras signaling in PTP1B-deficient fibroblasts
p120RasGAP expression and p62Dok phosphorylation are increased
Conclusions:
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ERK
1- Is receptor tyrosine phosphorylation increased?
3- Is Ras activated?
2- Is cell proliferation increased?
4. Is p120RasGAP activated?
5. Is p120RasGAP mRNA increased as well?
In PTPB1-/ - mice….
6. Is p120RasGAP 1 a substrate of PTP1B??
AKT
YESNO
NO
YESYES
Not directly