Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2α at the protein kinase C site but not at the protein kinase A-controlled site

Abstract

Streptozotocin-induced diabetes caused a profound increase in the steady-state level of phosphorylation of the α-subunit of the adenylate cyclase inhibitory protein Gi2 in hepatocytes. Unlike hepatocytes from control animals, those from streptozotocin-diabetic animals showed no increase in the phosphorylation of Gi2α in response to a challenge with the protein kinase C activator phorbol myristate acetate. However, a stimulatory effect of 8-bromo-cAMP on Gi2α phosphorylation was evident in hepatocytes from diabetic animals but this was severely reduced compared with that observed in hepatocytes from normal animals. Two-dimensional tryptic phosphopeptide mapping showed that Gi2α in resting hepatocytes from diabetic animals was phosphorylated exclusively at the protein kinase C site (C-site) but no labelling was evident at the protein kinase A-regulated site (AN-site). Treatment of hepatocytes from diabetic animals with phorbol myristate acetate did not change this pattern of labelling. In contrast, challenge of hepatocytes from diabetic animals with 8-bromo-cAMP led to the appearance of a new labelled phosphopeptide that was consistent with labelling at the AN-site. Analysis of the C-site and AN-site phosphopeptides from hepatocytes of diabetic animals treated with 8-bromo-cAMP showed that the increase in labelling of Gi2α caused by this ligand could be attributed almost entirely to labelling at the AN-site. Thus streptozotocin diabetes appears to cause enhanced labelling of hepatocyte Gi2α by exclusively increasing phosphorylation at the C-site. It is suggested that the increased labelling at the C-site reflects an augmentation of the protein kinase C signalling system in hepatocytes from streptozotocin-induced diabetic animals. This may have widespread functional consequences for these cells and may result either from an increased protein kinase C activity and/or a reduction in protein phosphatase 1 and/or 2A activity.