One of the most exciting advances in understanding the molecular actions of growth hormone (GH) was the identification of the tyrosine (Tyr) kinase JAK2 as a receptor-associated signaling molecule for the GH receptor (GHR). In this competing renewal we shall continue to test the hypothesis that activation of JAK2 in response to GH is a required initial step in GH signal transduction that results in the phosphorylation of multiple Tyr in GHR and JAK2, These phosphorylated Tyr serve as binding sites for molecules in various GH signaling pathways. During the past funding period, multiple signaling proteins and signaling pathways for OH were identified and/or characterized. Some of these signaling proteins were found to be activated as a consequence of binding to JAK2 (SH2-B, SIRPa) and others as a consequence of binding to GHR (SHP-2, Stat5). The docking site for others remains ambiguous (Shc, IRS, Stats 1 and 3). Efforts were initiated to identify the Tyr within GHR and JAK2 that are phosphorylated by JAK2, with 3 phosphorylated Tyr within JAK2 being identified.
In Aims land 2 of the current proposal, Tyr within JAK2 and GHR that are phosphorylated in response to OH will be identified and their regulation studied using a combination of 2 dimensional phosphopeptide mapping, phosphospecific antibodies and mass spectrometry.
Aim 3 will use microarray technology to identify populations of genes that are regulated by OH and determine whether expression of specific subpopulations of these genes require specific phosphorylated tyrosines in GHR and JAK2.
Aim 4 will determine which phosphorylated Tyr within GHR and JAK2 bind to known GH signaling proteins. Finally, Aim 5 Will use yeast 2-hybrid system to identify new signaling proteins that bind preferentially to activated, tyrosyl phosphorylated JAK2 and characterize their regulation by GH. To identify proteins that bind to specific phosphorylated tyrosines within GHR and JAK2, X expression libraries will be screened with phosphopeptides corresponding to phosphorylated Tyr containing sequences within GHR and JAK2. These studies will provide needed insight into: 1) the initiating steps in GHR signal transduction; 2) new signaling proteins and genes that are regulated by GH; 3) new signaling pathways utilized by OH; 4) the mechanisms by which these pathways are initiated; and 5) pathways that interact, either because they compete for the same binding site in GHR and/or JAK2, lie downstream of a common initial signaling molecule or require input form multiple upstream pathways. This insight should be of great assistance in delineating the molecular mechanisms by which OH elicits its diverse effects on growth and metabolism as well as in deciphering similarities and differences in the signaling pathways utilized by the cytokines that activate JAK kinases. Ultimately, it should facilitate the design the therapeutic agents that target specific growth and metabolic pathways regulated by GH and/or these other cytokines.
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