The overall goal is to elucidate signaling pathways from the insulin receptor. These proceed largely through the activation of protein kinases leading to changes in protein phosphorylation. One protein kinase that plays a prominent role in insulin signaling is Akt. Recently, we have discovered five novel insulin-elicited phosphoproteins in adipocytes, four of which are likely novel Akt substrates. In addition, recently a new method for identifying many sites of phosphorylation on many proteins in one sample by mass spectrometry has been developed. This method includes a procedure for quanitating the change in phosphorylation at each site upon treatment of the cell with an agent such as insulin.
Our specific aims, which build upon these advances, are: 1. To determine the cellular roles of the novel putative Akt substrates in insulin action. The initial focus will be a 250 kD protein (pp250) that contains a predicted GTPase activating protein (GAP) domain for the small G proteins Rap and Rheb. The GAP activity of recombinant pp250 toward Rap and Rheb, and the effect of phosphorylation of pp250 on the activity, will be determined. Depending upon whether pp250 is a GAP for Rap or Rheb, the effect of insulin on Rap or Rheb-regulated processes in adipocytes, and the role of pp250 in this effect, will be defined. 2. To identify other novel targets of insulin regulated phosphorylation in fat, muscle, and liver cells. Antibodies specific for phosphomotifs will be used to isolate groups of proteins that include ones phosphorylated on tyrosine by the insulin receptor and on serine/threonine by activated Akt and ERK. Novel sites of insulin-elicited phosphorylation will then be identified by the new phosphoproteomics methods. This research is directly relevant to diabetes. Detailed knowledge of insulin signaling provides a basis for understanding the changes that occur in insulin resistance and in insulin deficiency.
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