Substrate Capture Assay Shp1 In The Mapk Signaling Pathway In Target Proteins

SHP1, a kind of cytoplastic protein tyrosine phosphatase, is primarily expressed in hematopoietic cells and some epidermal cells. It was usually regarded as a negative regulator in cell growth, proliferation and differentiation in hematopoietic cells. However in some epidermal cells, the function of SHP1 is unclear. Some research results indicated that SHP1 can accelerate cell proliferation by activating the MAPK signal transduction pathway in these cells, but the detailed celluar and molecular mechanism is still unknown. One obstacle is to characterize the substrate(s) of SHP1 in the MAPK signal pathway. In this research,the substrate trapping technique was used to detect the physiological substrate(s) of SHP1 in the MAPK pathway,and a stable cell line expressing the CSDA substrate trapping mutant of SHP1-Flag was established.1. Using Substrate Trapping to detect the substrate of SHP1 in the MAPK pathwayIn order to uncover the substrate of SHP1 in the MAPK pathway, here we design a series of substrate trapping mutants of SHP1-Flag including SHP1CSDA-Flag, SHP1DAQA-Falg, SHP1TA-Flag. Serum-starved HEK293 cells transfected with the wild type SHP1-Flag or its mutants respectively were stimulated with EGF or not. The immunoprecipitates with anti-Flag were then subjected to SDS-PAGE and Western blot analysis with anti-phosphtyrosine antibody. It was found that in HEK293 cell, when activated by EGF, expression of the CSDA double mutant of SHP1-Flag resulted in hyperphosphorylation on tyrosine of a 90 KD around protein that was co-immunoprecipitated with the CSDA double mutant, but not with the wildtype. And this 90 KD protein was not found in the immunoprecipitates from cells expressing the wild type or the mutants of SHP1-Flag in the absence of EGF. Thus this 90 KD protein might be one substrate of SHP1-Flag in the MAPK pathway in HEK293 cell.2. Establishment of a stable cell line expressing SHP1CSDA-FlagAs shown, the CSDA double mutant of SHP1-Flag can trap the 90KD substrate of SHP1 in the MAPK pathway. Still, with a series of antibodies we can not identify this protein. So other means should be utilized to characterize this unknown protein. By using the Flp-In^TM system from Invitrogen corporation, a stable cell line expressing SHP1CSDA-Flag was established. Western blot analysis indicated that SHP1CSDA-Flag was highly expressed. Immunoblotting with anti-phosphotyrosine antibody from immunoprecipitates with anti-Flag also found the tyrosine hyperphosphorylated 90 KD protein associates with SHP1CSDA-Flag. With abundant culture of this cell line and the help of co-immunoprecipation to get enough 90 KD target protein, we hope to identify this 90KD protein by proteomics technology in the ongoing research.