Single substitution W593A dramatically decreases TEL-PDGFβR auto- and trans-kinase activity. (A) Autophosphorylation of TEL-PDGFβR and its mutants in Ba/F3 cells. Ba/F3 cells were lysed, and phosphorylated TEL-PDGFβR proteins were probed by using a specific mouse monoclonal antiphosphotyrosine antibody, 4G10. Ba/F3 cells transduced with empty retroviral vector were included as a control. The bottom panel shows similar levels of TEL-PDGFβR expression probed by polyclonal anti-PDGFβR antibody. (B) W593A mutant showed decreased transkinase activity in an in vitro kinase assay using MBP as an exogenous substrate. Following 4 hours serum-withdrawal treatment, TEL-PDGFβR immunocomplexes were isolated and incubated with substrate MBP as well as [γ-³²P] ATP. The ³²P-labeled proteins were visualized by autoradiography and phosphoimaging (top). As shown in the bottom panel, results of Coomassie blue staining revealed almost equal amounts of MBP used in each sample, and a parallel immunoprecipitation and Western blot assay was performed to confirm equal amounts of TEL-PDGFβR proteins in each kinase reaction. Wild-type TEL-PDGFβR is labeled as T/P wt, TEL-PDGFβR single mutants are labeled as W566A and W593A, whereas double mutant W566A/W593A is labeled as WW.