Angiotensin II Activates pp60 ^{c- src} in Vascular Smooth Muscle Cells

Abstract

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)

Not so strange bedfellows: G-protein-coupled receptors and Src family kinases

“…Early experiments demonstrated that Src, Fyn or Yes could be activated by a host of GPCRs, including the lysophosphatidic acid (LPA), a2A adrenergic, M1 muscarinic, M2 muscarinic, a-thrombin, endothelin, angiotensin II, bombesin, bradykinin, and V2 vasopressin receptors (Simonson and Herman, 1993;Chen et al, 1994;Ishida et al, 1995;Kramer et al, 1995;Linseman et al, 1995;Ptasznik et al, 1995;Luttrell et al, 1996;Rodriguez-Fernandez and Rozengurt, 1996;Schieffer et al, 1996;Simonson et al, 1996). From the outset, there was evidence for heterogeneity in the mechanisms underlying GPCR control of Src activity.…”

GIT1 Mediates Src-dependent Activation of Phospholipase Cγ by Angiotensin II and Epidermal Growth Factor

“…Activation of PLC␥ requires tyrosine phosphorylation as shown by inhibition of Ins(1,4,5)P 3 formation with inhibitors such as herbimycin A and genistein (13). A specific role for Src was suggested by demonstrating rapid activation of Src in VSMC, inhibition of PLC␥ activation with antibodies to Src, and impaired signal transduction in SrcϪ/Ϫ cells (12,16,23). It is well known that PLC␥1 and PLC␥2 are substrates for members of the Src kinase family (24,25).…”

Angiotensin II receptor pharmacology and AT1-receptor blockers

“…56,57 In smooth-muscle cells it has been shown that, for this purpose, the AT 1 receptor has to recruit the soluble tyrosine kinase pp60 c-src , since the receptor itself lacks intrinsic kinase activity. 58 PLC-g1 phosphorylation leads to the PIP 2 hydrolysis into 1,4,5-IP 3 and diacylglycerol. The induction of 1,4,5-IP 3 leads to intracellular calcium release, which in turn induces smooth-muscle cell contraction and other signaling events.…”