Download 3- Is Ras activated?

The role of protein tyrosine
phosphatase 1B in Ras signaling
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
Dubeet al. (2004) Proc. Natl. Acad. Sci. USA 101,
1834-1839
Protein Tyrosine phosphatases (PTPs)
Recall
• ~30 PTPs in human genome
• Remove phosphorylation
from tyrosine residues
Background
•>30 protein tyrosine kinases linked to malignant
transformation and cancer
•Overexpression and/or mutations
Thus, if down-regulate dephosphorylation eventspredict increased cancer
The question- If inhibit dephosphorylation, will
cancer increase??
Protein tyrosine phosphatase 1B (PTP1B)
•Negative regulator or several RTKs
• Removes phosphorylation from tyrosine residues
•A regulator of metabolism
•Reduces insulin, leptin and growth hormone
signaling
Background
Recall Receptor tyrosine kinases (RTKs)
•If inhibit PTP1B - Will cancer increase??
PTP1B
In PTPB1-/ - mice….
1- Is receptor tyrosine phosphorylation increased?
2- Is cell proliferation increased?
3- Is Ras activated?
ERK
AKT
4. Is p120RasGAP activated?
5. Is p120RasGAP mRNA increased as well?
6. Is p120RasGAP 1 a substrate of PTP1B??
Background
•PTPB1 deficient mice (PTPB1-/ -) are resistance to
diabetes and obesity
…But will PTPB1-/ - mice have increased tumor production?
Evidence
• IGF-1, EGF and PDGF receptor levels increase in PTPB1-/- mice
But… no increased tumorigenesis observed
Why the discrepancy??
Possibilities
•Cell-specific differences in RTKs?
•Tumorigenesis progression, but not initiation increased?
•PTP1B activates oncogenesis downstream of RTKs
Is tyrosine receptor phosphorylation increased in PTPB1-/ - mice?
WT KO
PDGF stimulation:
Fig. 1. Differential
regulation of PDGFsignaling pathways by
PTP1B
cellular tyrosine phosphorylation increased
ERK
AKT
Erk phosphorylation decreased
in PTP1B knockout cells
AKT phosphorylation
increased (more in PTP1B/- cells)
Thus, some proteims are (AKT), some aren’t (ERK)
Conclude- PTP1B seems be selective
Components of a Western analysis
Polyacrylamide gel
elecrophoresis,
transfer to nylon
membrane
1. Protein separation
2. Detection
Secondary Ab
(Anti-rabbit Ab)
Alkaline
peroxidase
Primary Ab
(Rabbit Anti-a1-antitrypsin)
A1-antitrypsin
a1-AT protein
2- Is cell proliferation increased?
Fig. 2. Decreased cell growth and Ras activity of SV40 TAg
transformed fibroblasts lacking PTP1B
1% FBS
10% FBS
decreased monolayer
growth in PTP1B-/- cells
WT
KO
WT KO
Answer- No, actually decreased.
3- Is Ras activated in PTPB1-/ - mice?
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
Answer- No, actually decreased activity.
What is not
changed in
PTPB1-/cells?
Answer- Expression levels of the
adapter proteins Shc or Grb2, or the
kinases (Src and Erk) (data not shown)
What is changed?
p120RasGAP
elevated…
p120RasGAP
decreased when
re-introduce
PTP1B
…but
p190RhoGAP
unchanged
…but no effect
on another
knockout cell
line
Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression
•Elevated in PTPB1-/- cells
•Normal levels when
introduce in PTPB1 gene
into cells
Is p120RasGAP mRNA increased as well?
Carry out RT-PCR reaction…
…then load product onto agarose gel
electrophoresis
p120RasGAP
mRNA is
elevated in
two
independent
cell lines
SV40-immortalized
Spontaneously
-immortalized
Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression
Conclude: PTPB1 suppresses RasGAP gene expression
Question- Is p120RasGAP 1 a substrate of PTP1B??
D181A mutant
GST Pulldown experiment
NIH3T3 cell
GST-tagged
PTP1B (WT or
D181A)
anti-pY
Cell lysate
Glutothione
GST-PTPB1
anti-p62Dok
Wash away unbound
Load bound onto gel
Use antibodies to detect
what binds PTPB1
Conclude- No, but p62Dok is.
Fig. 4. p62Dok is a putative substrate of PTP1B
Will Ras activation alone really cause cell
proliferation?
If introduce dominant
active Ras mutant
(V12Ras)….
Overcome Contact inhibition
Growth in Soft agar
….can transform
fibroblasts, even in
the absence of
PTP1B.
Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras
Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras
Overcome Contact inhibition
Mock
Growth in Soft agar
Conclusions:
p120RasGAP expression and
p62Dok phosphorylation are
increased
Fig. 6. Model of impaired Ras signaling in PTP1B-deficient fibroblasts
In PTPB1-/ - mice….
1- Is receptor tyrosine phosphorylation increased?
2- Is cell proliferation increased?
3- Is Ras activated?
YES
NO
NO
ERK
AKT
4. Is p120RasGAP activated?
YES
5. Is p120RasGAP mRNA increased as well?
YES
6. Is p120RasGAP 1 a substrate of PTP1B?? Not
directly