Activation of Extracellular Signal–Regulated Kinases (ERK1/2) by Angiotensin II Is Dependent on c-Src in Vascular Smooth Muscle Cells

Abstract

Among the angiotensin II (Ang II)-mediated signal events likely to be important in vascular smooth muscle cells (VSMCs) is activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The upstream mediators by which Ang II activates ERK1/2 remain poorly defined. Recently, we showed that Ang II activated c-Src, a nonreceptor kinase, which is a candidate to mediate Ang II signal events. To determine whether c-Src is required for ERK1/2 activation by Ang II, we studied the effects of Src family-selective tyrosine kinase inhibitors on ERK1/2 activation and also studied Ang II-mediated signal events in Src-deficient and Src-overexpressing VSMCs. The tyrosine kinase inhibitors, genistein and CP-188,556, blocked Ang II-mediated ERK1/2 activation in rat VSMCs (rVSMCs). We derived Src-deficient VSMCs from the aortas of c-Src knockout mice (Src-/- mVSMCs). Basal ERK1/2 activity was lower, and activation of ERK1/2 by Ang II was significantly decreased in Src-/- mVSMCs compared with wild-type mVSMCs, whereas ERK1/2 protein expression and ERK1/2 activation by phorbol 12-myristate 13-acetate were similar. To examine the role of c-Src further, we overexpressed wild-type or dominant-negative c-Src in rVSMCs using retroviral vectors. ERK1/2 activation by Ang II was significantly increased in rVSMCs that overexpressed c-Src, whereas ERK1/2 activation by Ang II was significantly inhibited in rVSMCs that overexpressed dominant-negative c-Src compared with control rVSMCs. These findings demonstrate that c-Src activation is required for Ang II stimulation of ERK1/2 in VSMCs and suggest an important role for c-Src in Ang II-mediated signal transduction.

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

“…These data support previous studies documenting an important role for Src-dependent tyrosine phosphorylation in PLC␥ activation and calcium mobilization in several cell types (50,54,55). In addition, results from our laboratory in SrcϪ/Ϫ cells and with c-Src inhibitors showed a correlation between decreased GIT1 tyrosine phosphorylation and activation of PLC␥ (23,26).…”

“…3E). These results are very similar to data previously published by our laboratory and others for protein phosphatase 1 in VSMC (23,26) and for protein phosphatase 2 in ECV304 cells (50), cardiac myocytes (51), and endothelial cells (52). We were unable to perform antisense GIT1 knockdown experiments in 293 cells because the endogenous level of GIT1 expression was too low to assay by Western blot (not shown).…”

“…Previous data from our laboratory suggested an essential role for c-Src in AT1R signal transduction, especially mediated by PLC␥, which is tyrosine-phosphorylated within 1 min of AngII binding (16,23,26,40). Therefore, we immunoprecipitated proteins from VSMC that associated with PLC␥ and characterized proteins that were dependent on Src using both pharmacologic inhibition and SrcϪ/Ϫ cells.…”

“…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).…”

Mechanism of Endothelin-1–Induced Contraction in Rabbit Basilar Artery

“…28 Angiotensin II stimulation was associated with a rapid activation of c-Src and activation of MAPK in vascular smooth muscle cells. 29 PI-3K activation may be upstream of Ras activation, and PI-3K may be involved in the function of Src, Shc, and Grb2. 30 It has been reported that the p110 subunit of PI-3K binds to an effector domain of Ras in vitro, 30 and the PI-3K inhibitor wortmannin reduced insulin-induced MAPK activity.…”