In vitro characterization ofp90 ribosomal S6 kinase domains and in vivo analysis of insulin receptor substrate-2 hepatic function

Signal transduction is the mechanism by which cells communicate and respond to their environment. The work presented in this thesis used both in vitro and in vivo approaches to elucidate the regulation and function of specific signaling molecules. Part one of the thesis explores p90 ribosomal S6 kinase (RSK) catalytic activity while part two addresses the feasibility of adenovirus-mediated gene replacement therapy in a murine model of type 2 diabetes mellitus.; RSK activity has been implicated downstream of the Ras-MAPK (mitogen-activated protein kinase) pathway which when unregulated commonly leads to cellular transformation and oncogenesis. The RSK amino acid sequence contains two putative kinase domains. Part one of the thesis was designed to identify the activities and regulation of these two potential catalytic domains. The hypothesis that the amino-terminal kinase domain of RSK was responsible for in vitro -identified substrate phosphotransferase activity was confirmed using analysis of transiently expressed RSK mutant proteins in intact cells. In vitro studies employing recombinant proteins revealed a carboxyl-terminal autophosphotransferase activity that was further stimulated by activated MAPK phosphorylation.; The insulin receptor substrate (IRS)-2 protein is required for the maintenance of glucose homeostasis. The Irs2−/− mice have defects in both insulin action and production leading to severe hyperglycemia and development of type 2 diabetes mellitus. The liver is a major site of carbohydrate metabolism that is adversely impacted by Irs2 deletion. The hypothesis in part two of the thesis postulated that adenovirus (Ad)-mediated replacement of IRS-2 expression in liver of the Irs2−/− mice would contribute to a normalization of hepatic glucose production and a delay or abrogation of overt diabetes development. The data described herein indicated Ad-IRS2 administration failed to impede diabetic disease progression in the Irs2−/− mouse. Additional results from this work suggested over-expression of hepatic IRS-2 leads to excessive intrahepatocyte glycogen and lipid accumulation due in part to alterations in specific enzymatic activities involved in the insulin-stimulated glycogen synthesis pathway.