Endothelin-1 induces serine phosphorylation of the adaptor protein p66Shc and its association with 14-3-3 protein in glomerular mesangial cells.
Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC). In the current study, it is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66(Shc)). ET-1-induced serine phosphorylation of p66(Shc) requires activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling module and is efficiently inhibited by both a MAPK/ERK kinase (MEK)-selective inhibitor and adenovirus-mediated transfer of a dominant interfering MEK1 mutant. Furthermore, adenovirus-mediated transfer of a constitutively active MEK1 mutant was found to markedly increase p66(Shc) serine phosphorylation. Adenoviruses encoding constitutively active mutants of MAPK kinases 3 and 6 (upstream kinases of p38(MAPK)) and 7 (upstream kinase of c-Jun NH(2)-terminal kinase) failed to induce serine phosphorylation of this adaptor protein. Serine phosphorylation of p66(Shc) resulted in its association with the serine binding motif-containing protein 14-3-3. ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66(Shc) was confirmed in experiments employing anti-phospho-14-3-3 binding motif antibodies. These studies are the first to demonstrate that G protein-coupled receptors stimulate serine phosphorylation of p66(Shc) and the first to report the formation of a signaling complex between p66(Shc) and 14-3-3.
Foschi
M
M
Department of Internal Medicine, University of Florence, v. le Morgagni 85, Florence 50141, Italy. m.foschi@dfc.unifi.it
Franchi
F
F
Han
J
J
La Villa
G
G
Sorokin
A
A
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D019332:Endothelin-1; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D012694:Serine; D014446:Tyrosine 3-Monooxygenase