Endothelin-1 Induces Serine Phosphorylation of the Adaptor Protein p66Shc and Its Association with 14-3-3 Protein in Glomerular Mesangial Cells

Marco Foschi,Franco Franchi,Jiahuai Han,Giorgio La Villa,Andrey Sorokin

doi:10.1074/jbc.m102008200

Abstract

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.

Highlights

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC)
The adaptor protein Shc, one of the major tyrosine-phosphorylated proteins in growth factor-stimulated cells, has been demonstrated to link tyrosine kinase receptors to the activation of mitogen-activated protein kinase (MAPK) cascades [33]
We and others have produced evidence that the recruitment of Shc is required for G protein-coupled receptor-dependent Ras-Raf-MAPK/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway activation [13, 43]

Summary

Mesangial Cells*

It is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66Shc). ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66Shc 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 p66Shc and the first to report the formation of a signaling complex between p66Shc and 14-3-3. Phosphorylation of tyrosine, serine, and threonine residues in target molecules generates recognition motifs for a number of protein-protein interaction domains, thereby promoting the formation of multisubunit signaling complexes and constituting the basis of a complicated network of intracellular signaling pathways [4]. Evidence is provided that ET-1-induced serine phosphorylation of p66Shc generates a serine recognition motif that may be responsible for the direct association of p66Shc with 14-3-3

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DISCUSSION