The angiotensin II type-1 (AT(1)) receptor, a G protein-coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-gamma 1 and to be activated by G protein-coupled receptors. We hypothesized that pp60(c-src) associates with the AT(1) receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60(c-src) from Ang II-stimulated vascular smooth muscle cells and measured pp60(c-src) activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60(c-src) activity within 1 minute. A similar increase in Ang II-stimulated pp60(c-src) activity was observed in Chinese hamster ovary cells transfected with the AT, receptor but not in untransfected cells. These data are the first to show that pp60(c-src) is activated by Ang II. To determine if pp60(c-src) associated with the AT, receptor, the AT, receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60(c-src) antibodies. No pp60(c-src) was detected. In addition, direct interaction between the AT(1) receptor and pp60(c-src) could not be demonstrated by using a glutathione S-transferase (GST)-AT(1) fusion protein to bind proteins from cell lysates stimulated by Ang II. In combination with recent findings that anti-pp60(c-src) antibodies inhibit Ang II-mediated PLC-yl phosphorylation, our data suggest an important role for pp60(c-src) in Ang II signal transduction.
