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

Protein Tyrosine phosphatases (PTPs)

• ~30 PTPs in human genome

• Remove phosphorylation from tyrosine residues

Recall

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

PTP1B

Recall Receptor tyrosine kinases (RTKs)

Background

•If inhibit PTP1B - Will cancer increase??

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

•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

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

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

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

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.

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

•Elevated in PTPB1-/- cells

•Normal levels when introduce in PTPB1 gene into cells

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

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

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?

Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras

Growth in Soft agar

Overcome Contact inhibition

Mock

Fig. 6. Model of impaired Ras signaling in PTP1B-deficient fibroblasts

p120RasGAP expression and p62Dok phosphorylation are increased

Conclusions:

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