1 / 18

Nadia Dubé * , Alan Cheng * and Michel L. Tremblay

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.

ita
Download Presentation

Nadia Dubé * , Alan Cheng * and Michel L. Tremblay

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. Protein Tyrosine phosphatases (PTPs) Recall • ~30 PTPs in human genome • Remove phosphorylation from tyrosine residues

  3. Background • >30 proteintyrosine kinases linked to malignant transformationand cancer • Overexpression and/or mutations Thus, if down-regulate dephosphorylation events- predict 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

  4. Background Recall Receptor tyrosine kinases (RTKs) • If inhibit PTP1B - Will cancer increase?? PTP1B

  5. In PTPB1-/ - mice…. 1- Is receptor tyrosine phosphorylation increased? 2- Is cell proliferation increased? 3- Is Ras activated? ERK AKT 4. Is p120RasGAPactivated? 5. Is p120RasGAPmRNA increased as well? 6. Is p120RasGAP 1 a substrate of PTP1B??

  6. 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

  7. Is tyrosine receptor phosphorylation increased in PTPB1-/ - mice? WT KO Fig. 1. Differential regulation of PDGF-signaling pathways by PTP1B PDGF stimulation: cellular tyrosine phosphorylation increased 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

  8. Components of a Western analysis Polyacrylamide gel elecrophoresis, transfer to nylon membrane 1. Protein separation Alkaline peroxidase 2. Detection Primary Ab (Rabbit Anti-a1-antitrypsin) Secondary Ab (Anti-rabbit Ab) A1-antitrypsin a1-AT protein

  9. 2- Is cell proliferation increased? Fig. 2. Decreased cell growth and Ras activity of SV40 TAg transformed fibroblasts lacking PTP1B 10% FBS 1% FBS decreased monolayer growth inPTP1B-/- cells Cell proliferation KO WT KO WT Answer- No, actually decreased.

  10. Fig. 2. Decreased cell growth and Ras activity of SV40 TAg transformed fibroblasts lacking PTP1B 3- Is Ras activated in PTPB1-/ - mice? diminished Ras-GTP levels In PTP1B-/- cells PDGFR expression is increased in PTP1B-/- cells Answer- No, actually decreased activity.

  11. What is not changed in PTPB1-/- cells? Answer- Expression levels of the adapterproteins Shc or Grb2, or the kinases (Src and Erk) (data not shown) What is changed? …but p190RhoGAP unchanged p120RasGAP elevated… …but no effect on another knockout cell line p120RasGAP decreased when re-introduce PTP1B Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression

  12. Elevated in PTPB1-/- cells • Normal levels when introduce in PTPB1 gene into cells

  13. Is p120RasGAPmRNA increased as well? Carry out RT-PCR reaction… …then load product onto agarose gel electrophoresis p120RasGAP mRNA is elevated in two independent cell lines Spontaneously -immortalized SV40-immortalized Fig. 3. PTP1B-deficient cells display increased p120RasGAP expression Conclude: PTPB1 suppresses RasGAP gene expression

  14. Question- Is p120RasGAP1 a substrate of PTP1B?? D181A mutant NIH3T3 cell GST-tagged PTP1B (WT or D181A) anti-pY Cell lysate GST-PTPB1 Glutothione anti-p62Dok Wash away unbound Load bound onto gel Use antibodies to detect what binds PTPB1 GST Pulldown experiment Conclude- No, but p62Dok is. Fig. 4. p62Dok is a putative substrate of PTP1B

  15. Will Ras activation alone really cause cell proliferation? If introduce dominant active Ras mutant (V12Ras)…. Overcome Contact inhibition Two properties of transformed cells ….can transform fibroblasts, even inthe absence of PTP1B. Growth in Soft agar Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras

  16. Fig. 5. Transformation of PTP1B-deficient fibroblasts by activated Ras Overcome Contact inhibition Mock Growth in Soft agar

  17. Conclusions: p120RasGAP expression and p62Dok phosphorylationare increased Fig. 6. Model of impaired Ras signaling in PTP1B-deficient fibroblasts

  18. In PTPB1-/ - mice…. YES 1- Is receptor tyrosine phosphorylation increased? NO 2- Is cell proliferation increased? 3- Is Ras activated? NO ERK AKT YES 4. Is p120RasGAPactivated? YES 5. Is p120RasGAPmRNA increased as well? Not directly 6. Is p120RasGAP 1 a substrate of PTP1B??

More Related