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,

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

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% FBS 1% FBS 2- Is cell proliferation increased? Answer- No, actually decreased. KO WT Cell proliferation

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-immortalized Spontaneously -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-PTPB1 Glutothione 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. Two properties of transformed cells 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 YES NO YES Not directly