title: Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2 alpha 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 alpha-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 Gi2 alpha in response to a challenge with the protein kinase C activator phorbol myristate acetate. However, a stimulatory effect of 8-bromo-cAMP on Gi2 alpha 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 Gi2 alpha 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 Gi2 alpha 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 Gi2 alpha 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 wide-spread 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.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
authors.forename: N J
authors.initials: NJ
authors.lastname: Morris
authors.forename: M
authors.initials: M
authors.lastname: Bushfield
authors.forename: M D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D011518:Proto-Oncogene Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; D013755:Tetradecanoylphorbol Acetate