Biphasic activation of p21(ras) by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21(ras) activation. Whereas p21(ras) activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21(ras) activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21(ras) in rat glomerular mesangial cells. The first peak of activation of p21(ras), at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21(ras) results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21(ras) activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21(ras) and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21(ras) at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21(ras)-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21(ras) activation causes sequential stimulation of divergent downstream signaling pathways.