Podocyte Protein, Nephrin, Is a Substrate of Protein Tyrosine Phosphatase 1B
Figure 5
PTP-PEST decreases nephrin phosphorylation via Src-family kinase. (a) Pull-down was performed as in Figure 4(a) with additional plasmids of GST-PTP-PEST-WT and GST-PTP-PEST-CS (substrate-trapping mutant). The substrate-trapping mutant of PTP1B, but not of PTP-PEST, pulled down nephrin. (b) Cos-1 cells were transiently cotransfected with pEBG (empty vector), GST-PTP-PEST-WT, or GST-PTP-PEST-CS (substrate trapping mutant) and fyn. After 24 hrs, cell lysates were subjected to GST pull down as in Figure 4(a) or to immunoprecipitation for fyn. Only the substrate-trapping mutant of PTP-PEST (CS), but not the wild-type, pulled down fyn or was coimmunoprecipitated with fyn. fyn, which was pulled down by PTP-PEST-CS, was strongly phosphorylated at pY418, suggesting that pY418 containing region is the substrate of PTP-PEST. Wild-type PTP-PEST decreased pY418-Src in total lysates. Note that pY418 antibody cross-reacts to all the Src-family kinases.
