Biphasic activation of p21ras 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 p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras 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 p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, 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 p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras 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 p21ras 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 p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.
Foschi
M
M
Department of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee 53226, USA.
Chari
S
S
Dunn
M J
MJ
Sorokin
A
A
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D054638:Dual Specificity Phosphatase 1; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D051380:GRB2 Adaptor Protein; D005920:Glomerular Mesangium / Q000187:drug effects / Q000201:enzymology; D020662:Guanine Nucleotide Exchange Factors; D017874:Immediate-Early Proteins / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017466:Receptors, Endothelin / Q000187:drug effects* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020663:ras Guanine Nucleotide Exchange Factors
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018797:Cell Cycle Proteins; D019332:Endothelin-1; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D017874:Immediate-Early Proteins; D011506:Proteins; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020663:ras Guanine Nucleotide Exchange Factors; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases; D016283:Proto-Oncogene Proteins p21(ras)