Growth hormone (GH) is a powerful growth-promoting and metabolic hormone whose mechanism(s) of action is only partially understood. GH triggers several intracellular signaling pathways by dimerizing the transmembrane GH receptor (GHR) and activating the GHR-associated JAK2 tyrosine kinase. Our previous work revealed GHR and JAK2 regions that allow GH-induced activation of JAK2 and a downstream effector of GH signaling, STAT5b. Another pathway engaged by GH activates the extracellular signal-regulated kinase (ERK) group of MAP kinases. MAPK (ERK 1 and 2) activation regulates various aspects of cell behavior and gene expression. Yet how GH-induced JAK2 activation promotes MAPK activation, the degree of overlap with STAT pathway activation, and biological consequences of GH-induced MAPK activation are unclear. Our recent observations regarding mechanisms of GH-induced MAPK activation and its signaling relevance include: 1) OH-induced MAPK activation is augmented by insulin receptor substrate-1 (IRS-1) and this augmentation may be facilitated by physical interaction of IRS-1 with JAK2; 2) GH promotes tyrosine phosphorylation of the protein tyrosine phosphatase, SHP-2, which positively regulates OH-induced c-fos transcription and may thus positively regulate GH-induced MAPK signaling; 3) OH causes tyrosine phosphorylation of Gab 1, an adaptor protein important in growth factor-induced MAPK signaling; 4) GH, via a MEK1/MAPK-dependent pathway, inhibits activation of ErbB-2, an epidermal growth factor (EGF) receptor family member, and causes desensitization of EGF signaling. We hypothesize: 1) GHR and JAK2 association with particular signaling molecules allows GH-induced access to its signaling pathways, including the MAPK pathway; 2) OH, by accessing particular pathways or interactions, modulates signaling by other growth factors.
Our specific aims are: 1. Define regions of JAK2 necessary for its GH-induced activation and for IRS-1 association and activation by reconstituting a JAK2-deficient cell line with JAK2 (wild-type and mutants) and IRS-1. We will perform coimmunoprecipitation and in vitro association studies and assay JAK2 activation, IRS-1 tyrosine phosphorylation, and downstream signals. 2. Define mechanisms by which IRS-1, SRP-2, Gab1, and EGFR may be involved in GH-induced MAPK activation. Biochemical analysis will focus on CHO-rbGHR cells, 32D-rbGHR cells, and SHP-2-deficient fibroblasts and we will develop an inducible expression system to test MAPK activators in 3T3-F442A cells. We will test the impact of these molecules on GH-induced IGF-1 gene expression in a C6 glioma cell transfection system. 3. Investigate the basis for MEK1/MAPK-dependent GR-induced desensitization of ErbB-2 signaling. Roles of EGFR, MAP kinase activators, and selected OH-activated serine/threonine kinases and phosphatases will be studied and sites of OH-induced phosphorylation in the ErbB-2 cytoplasmic domain will be determined. Results of these studies will significantly expand our knowledge of GH and cytokine signaling and of biologically relevant crosstalk between cytokine and growth factor signaling systems.
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