Multi-Site Phosphorylation of Hepatocyte Gi-2

Morris, Nicholas James (1994) Multi-Site Phosphorylation of Hepatocyte Gi-2. PhD thesis, University of Glasgow.

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The phosphorylation of the heterotrimeric guanine nucleotide binding proteins has received increasing attention as a possible mode of controlling intracellular signalling. Here further evidence is submitted which helps to confirm the occurrence of such phosphorylation events in rat hepatocytes, to identify the phosphorylation site in alpha-Gi-2, to examine the effects of insulin on the phosphorylation and to investigate any changes in the phosphorylation characteristics in animal models of type I and type II diabetes. Using the antiserum 1867 it was possible to immunoprecipitate specifically the phosphorylated form of guanine nucleotide binding protein alpha-Gi-2 from rat hepatocytes. By carrying out two dimensional phosphopeptide mapping and phosphoamino acid analysis of the protein it was found that phosphorylation occurred at two different serines and that one of the sites was most probably a protein kinase C (PKC) phosphorylation site whilst the other, although being protein kinase A (PICA)-dependent, was phosphorylated by an unknown kinase. By the use of the enzymes trypsin and Staphylococcus aureus V8, and theoretical analysis of possible phosphopeptides and their migration in the two-dimensional chromatography, it was possible to predict the phosphorylation site for the PKC-mediated phosphorylation as either serine 47 or serine 144, with serine 144 being the most likely candidate. The identification of the other phosphorylation site was not possible from the data produced. These sites were also found to occur in alpha-Gi-2 from hepatocytes in streptozotocin-treated Sprague Dawley rats and in both lean and obese Zucker rats. The incubation of hepatocytes from Sprague Dawley rats with insulin produced both a time and concentration dependent inhibition of the 32P-labelling of alpha-Gi-2 with the inhibition becoming significant after 2 minutes and insulin being effective at 1 nM. As it is known that the phosphorylation of alpha-Gi-2 in rat hepatocytes is under the control of a futile cycle the point of action of insulin was investigated by the use of a range of ligands that interacted with the inositol phosphate and cAMP signalling pathways and hence stimulated the phosphorylation of alpha-Gi-2. From these studies it was found that insulin was able to inhibit some of the ligand-induced phosphorylation of alpha-Gi-2 and this indicated that insulin interacted with at least two components of the cycle, that is a kinase and a phosphatase. Interestingly, insulin also enhanced the phosphorylation induced by glucagon and the insulin inhibition of phosphorylation was reversed by amylin which also caused a cAMP-dependent phosphorylation. Evidence was also found that a cAMP-dependent phosphorylation of alpha-Gi-2 may occur even when the cAMP system is not being stimulated. The induction of type I diabetes in Sprague Dawley rats with streptozotocin caused a loss of the insulin-induced inhibition of alpha-Gi-2 phosphorylation and also reduced the levels of phosphorylation of alpha-Gi-2 achieved by 8-bromo-cAMP, PMA and glucagon and abolished the insulin enhancement of glucagon stimulated phosphorylation. In the obese Zucker rat model of type II diabetes and their lean litter mates, insulin did not effect the phosphorylation of alpha-Gi-2 and the enhancement of glucagon stimulated phosphorylations was lost. The compound BRL 49653, which normalises glucose handling in animal models of diabetes, did not effect the ligand induced phosphorylations of alpha-Gi-2 other than abolishing the significant insulin inhibition of glucagon stimulated phosphorylation in the lean animals. In conclusion, the activated insulin receptor does interact with alpha-Gi-2 and although the result of this interaction on cellular signalling is not known it does seem likely that it is not involved in the control of glucose homeostasis but some other aspect of insulin signalling.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Miles Housley
Keywords: Biochemistry
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75973
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:09
Last Modified: 19 Nov 2019 17:09

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