Angiotensin II activates pp60c-src in vascular smooth muscle cells.
The angiotensin II type-1 (AT1) 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 pp60c-src associates with the AT1 receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II-stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II-stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1 receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1 receptor, the AT1 receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1 receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1 fusion protein to bind proteins from cell lysates stimulated by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishida
M
M
Division of Cardiology, University of Washington, Seattle 98195, USA.
Marrero
M B
MB
Schieffer
B
B
Ishida
T
T
Bernstein
K E
KE
Berk
B C
BC
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000378:metabolism; D015398:Signal Transduction; D013268:Stimulation, Chemical; D013997:Time Factors; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D011945:Receptors, Angiotensin; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D005982:Glutathione Transferase; D016392:Proto-Oncogene Proteins pp60(c-src); D010751:Phosphopyruvate Hydratase