Endostatin-induced tyrosine kinase activity in complex with Shb SH2 domain.

(A) IBE or PAE/FGFR-1 cells were incubated with (+) or without (−) FGF-2 and endostatin for 10 minutes. Cell lysates were incubated with the GST Shb SH2 domain fusion protein, and associated proteins were analyzed by SDS-PAGE and immunblotting with phosphotyrosine antibodies (blot PY) or by incubation in the presence of γ³²P]ATP (kinase assay). The migration rate of the Shb SH2-associated protein of 125 kd is indicated to the right. (B) PAE cells or Swiss 3T3 cells were incubated with indicated concentrations of endostatin and processed for immunoprecipitation using antiphosphotyrosine antibodies 4G10 (IP PY) and in vitro kinase assay. PAE cells treated with the endostatin mutant R158/270A were analyzed similarly. (C) PAE cells were incubated with (+) or without (−) FGF-2 and endostatin for 10 minutes, lysed, and similar protein amounts from the different samples were separated by SDS-PAGE, transferred to nitrocellulose and blotted using antiphosphotyrosine antibodies (4G10). Migration rates of marker proteins are indicated to the right. Phosphotyrosyl proteins induced by endostatin are indicated by *.