Angiotensin II Stimulates Tyrosine Phosphorylation of Phospholipase C-γ–Associated Proteins

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Abstract
Stimulation of phospholipase C-γ (PLC-γ) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-γ via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-γ, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-γ–associated proteins, RASM cells were labeled with [ 32 P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-γ was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-γ–associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src–transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-γ–associated protein that may be required for Ang II–mediated signal transduction.