The Effect of Protein Kinase-C Inhibition on Insulin Receptor Phosphorylation

Abstract

The effect of protein kinase-C (PKC) inhibition on insulin receptor phosphorylation in HepG2 cells was analyzed by two-dimensional tryptic phosphopeptide maps. In basal cells, there was one major insulin receptor-derived tryptic phosphothreonine peptide and at least four phosphoserine peptides. Phorbol 12,13-dibutyrate (PDBU) stimulated phosphorylation of the phosphothreonine peptide, some of the basal phosphoserine peptides, and at least one phosphoserine peptide that was not detected in the basal state. Staurosporine completely inhibited the PDBU-mediated phosphorylation. Although staurosporine also inhibited basal phosphorylation of the phosphothreonine peptide, down-regulation of PKC did not, suggesting that PKC does not mediate basal insulin receptor phosphorylation. Insulin treatment resulted in the appearance of four phosphotyrosine peptides. It also stimulated the phosphorylation of at least two phosphoserine peptides. One of these may have been a complex of two or more distinct but poorly resolved phosphopeptides, which was seen in basal cells and a component of which seemed to be stimulated by PDBU. However, neither staurosporine nor down-regulation of PKC diminished insulin-stimulated serine phosphorylation of these peptides, indicating that insulin-stimulated receptor serine phosphorylation did not involve PKC activity. The addition of staurosporine to cells that had been incubated with PDBU resulted in the very rapid decay of phosphorylation of the phosphothreonine-containing peptide, indicating that this site of phosphorylation turns over very rapidly, while some of the other phosphoserine-containing peptides, including the major unique site of phosphorylation stimulated by PDBU, turned over more slowly. Thus, the insulin receptor contains several sites of serine/threonine phosphorylation, some of which are substrates for more than one protein kinase. This may permit complex modulation of insulin receptor functions in response to multiple signalling pathways.