Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma-associated proteins. Characterization of a c-Src-dependent 97-kD protein in vascular smooth muscle cells.
Stimulation of phospholipase C-gamma (PLC-gamma) 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-gamma via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-gamma, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-gamma-associated proteins, RASM cells were labeled with [32P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-gamma 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-gamma-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-gamma-associated protein that may be required for Ang II-mediated signal transduction.
Schmitz
U
U
Department of Medicine, University of Washington, Seattle 98195-7710, USA.
Ishida
M
M
Berk
B C
BC
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D016391:Genes, src; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D000804:Angiotensin II; D014443:Tyrosine; D019061:src-Family Kinases; D010738:Type C Phospholipases