Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Adaptor Proteins, Vesicular Transport*
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Animals
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Cell Cycle Proteins*
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Cells, Cultured
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Dual Specificity Phosphatase 1
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Endothelin-1 / pharmacology*
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Enzyme Activation / drug effects
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GRB2 Adaptor Protein
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Glomerular Mesangium / drug effects
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Glomerular Mesangium / enzymology
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Guanine Nucleotide Exchange Factors
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Immediate-Early Proteins / metabolism
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Kinetics
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Male
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Models, Biological
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Phosphatidylinositol 3-Kinases / metabolism*
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Phosphoprotein Phosphatases*
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Phosphorylation
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Protein Phosphatase 1
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Protein Processing, Post-Translational
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Protein Tyrosine Phosphatases / metabolism
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Proteins / metabolism
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Proto-Oncogene Proteins p21(ras) / metabolism*
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Rats
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Rats, Sprague-Dawley
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Receptors, Endothelin / drug effects*
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Receptors, Endothelin / physiology
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Recombinant Fusion Proteins / metabolism
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Shc Signaling Adaptor Proteins
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Signal Transduction / drug effects*
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Src Homology 2 Domain-Containing, Transforming Protein 1
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ras Guanine Nucleotide Exchange Factors
Substances
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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Cell Cycle Proteins
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Endothelin-1
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GRB2 Adaptor Protein
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Grb2 protein, rat
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Guanine Nucleotide Exchange Factors
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Immediate-Early Proteins
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Proteins
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Receptors, Endothelin
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Recombinant Fusion Proteins
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Shc Signaling Adaptor Proteins
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Shc1 protein, rat
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Src Homology 2 Domain-Containing, Transforming Protein 1
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ras Guanine Nucleotide Exchange Factors
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Phosphatidylinositol 3-Kinases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1
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Dual Specificity Phosphatase 1
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Dusp1 protein, rat
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Protein Tyrosine Phosphatases
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Proto-Oncogene Proteins p21(ras)