Protein kinase C-ζ phosphorylates serine/threonine residues at the C-terminal binding motif of the tyrosine phosphatase SHP-2 of insulin receptor substrate 1

The phosphorylation of serine-/threonine-residues of insulin receptor substrate-1 (IRS-1) by protein kinase C-ζ (PKC-ζ), which leads to an increased tyrosine dephosphorylation of IRS-1, has been recently identified as a novel hyperinsulinemia-induced negative feed-back regulation of the insulin signalling pathway. To identify the underlying molecular mechanism, we investigated the serine-/threonine-residues in IRS-1 phosphorylated by PKC-ζ. Because the activity of the tyrosine phosphatase SHP-2, which dephosphorylates IRS-1, is regulated by the binding to the C-terminal part of IRS-1 (IRS-1^c) this region was studied. In the in vitro assay the atypical PKC ζ phosphorylates IRS-1^c comparable to PKC-θ and -β<INF>1</INF>, whereas PKC-β<INF>2</INF> showed lower and PKC-i and PKC-α showed negligible phosphorylation. We observed one major ³²P-motif labelled by PKC-ζ, containing four phosphorylated serine/threonine residues (Ser 1215, 1216, 1220 and Thr 1221) near the C-terminal end of IRS-1. These sites are closely adjacent to tyrosine 1222, which represents one of the two binding sites for the protein tyrosine phosphatase SHP-2. In conclusion, the PKC-ζ-dependent in vitro phosphorylation of IRS-1 at a site closely situated to a crucial binding site of the tyrosine phosphatase SHP-2 suggests the modulation of the dephosphorylation activity of SHP-2 by PKC-ζ. Furthermore, our findings can contribute to the explanation of the complex positive and negative regulatory action of PKC-ζ on insulin signalling.