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The effect of protein kinase-C inhibition on insulin receptor phosphorylation
1Department of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
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.
Insights
Protein kinase-C (PKC) inhibition affects insulin receptor phosphorylation. While PKC mediates some basal and phorbol ester-stimulated phosphorylation, it does not appear to regulate insulin-stimulated serine phosphorylation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- The insulin receptor is a key regulator of glucose metabolism.
- Insulin receptor phosphorylation, particularly on serine residues, plays a crucial role in modulating its activity.
- Protein kinase-C (PKC) is implicated in various cellular signaling pathways, but its specific role in insulin receptor phosphorylation requires further elucidation.
Purpose of the Study:
- To investigate the effect of protein kinase-C (PKC) inhibition on insulin receptor phosphorylation in HepG2 cells.
- To differentiate the roles of PKC in basal, phorbol ester-stimulated, and insulin-stimulated insulin receptor phosphorylation.
- To identify specific phosphorylation sites and their turnover rates in response to different stimuli.
Main Methods:
- Analysis of insulin receptor phosphorylation using two-dimensional tryptic phosphopeptide maps in HepG2 cells.
- Treatment with phorbol 12,13-dibutyrate (PDBU) to activate PKC.
- Inhibition of PKC using staurosporine and down-regulation of PKC.
- Stimulation of cells with insulin to assess insulin-specific phosphorylation events.
Main Results:
- Basal insulin receptor phosphorylation included phosphothreonine and phosphoserine peptides.
- Phorbol 12,13-dibutyrate (PDBU) stimulated phosphorylation of specific phosphothreonine and phosphoserine sites, which was inhibited by staurosporine.
- Insulin treatment induced phosphotyrosine peptides and stimulated specific phosphoserine phosphorylation, independent of PKC activity.
- PKC inhibition by staurosporine rapidly affected phosphothreonine peptide phosphorylation, while other sites showed slower turnover.
Conclusions:
- The insulin receptor possesses multiple serine/threonine phosphorylation sites.
- PKC mediates certain basal and PDBU-stimulated phosphorylation events on the insulin receptor.
- Insulin-stimulated serine phosphorylation of the insulin receptor does not involve PKC activity.
- Differential turnover rates of phosphorylation sites suggest complex regulatory mechanisms modulating insulin receptor function.