Molecular Mechanisms of Growth Hormone-Induced Signal Transduction and SH2B1

Abstract

Growth hormone (GH) regulates overall body growth and metabolism and is used therapeutically for a variety of clinical applications. GH binding to its receptor activates the tyrosine kinase, JAK2. Active JAK2 initiates multiple cellular responses to GH, including regulation of the cytoskeleton, that lead to cellular proliferation, differentiation and migration. Recent studies questioned whether JAK2 is the primary kinase responsible for transducing all GH signals. This thesis establishes that JAK2 is the primary kinase responsible for GH-mediated activation of Stat1, Stat3, Stat5, ERK1/2 and Akt in two established models of GH signaling, 3T3-F442A preadipocytes and H4IIE hepatoma cells. This work also characterizes two novel mechanisms by which the adaptor protein, SH2B1, may modulate GH-induced regulation of the actin cytoskeleton. The cytoskeleton scaffolding protein, II-spectrin, was identified as an SH2B1 interacting protein. JAK2 was found to phosphorylate II-spectrin in an SH2B1-dependent manner. GH induced formation of a II-spectrin/SH2B1/JAK2 complex in 3T3-F442A cells and induced a shift in II-spectrin subcellular localization in H4IIE cells. These results suggest that GH may regulate the cytoskeleton through an SH2B1/II-spectrin interaction. SH2B1 was also found to localize to focal adhesions, which are cytoskeletal structures that regulate cell anchorage and motility. GH increased the dynamic cycling of SH2B1 into and out of focal adhesions. In addition, PMA induced redistribution of SH2B1 out of focal adhesions. Two serines within SH2B1 (serines 161 and 165) were implicated in regulating this PMA effect as well as the dynamic cycling of SH2B1 into and out of focal adhesions. Mutation of SH2B1 serine 165 to glutamate also increased the overall focal adhesion number in cells. These results implicate SH2B1 as a novel focal adhesion protein and suggest that stimuli that induce phosphorylation of SH2B1 at serines 161 and/or 165 regulate SH2B1 dynamics at focal adhesions and may contribute to the regulation of overall focal adhesion number. Taken together, the work in this thesis demonstrates that GH activates downstream signaling through JAK2 in 3T3-F442A and H4IIE cells. This work also provides evidence that GH regulates the cytoskeleton through an SH2B1/II-spectrin interaction and/or modulating SH2B1 at focal adhesions.