Identification of Steroid-Sensitive Gene-1 as a Novel JAK2 Binding Protein and the Physiological Consequences of this Interaction.

Abstract

Growth hormone (GH) is an important regulator of body growth and metabolism. GH binding to its receptor activates the receptor-associated tyrosine kinase, JAK2, which in turn, initiates multiple signaling cascades, including activation of Stats 1, 3, 5a, and 5b. JAK2 is an important signaling molecule activated by GH. However, very few proteins have been identified as binding partners for active JAK2. We identified steroid-sensitive gene-1 (SSG1) as a JAK2 binding protein that binds to the active form of JAK2 in a yeast two-hybrid screen of a rat adipocyte cDNA library. Full-length SSG1 binds to the kinase-active form of JAK2 and its kinase (JH1) domain, but not kinase-inactive JAK2 (K882E) or JH1 (K882E). Full-length SSG1 binds JAK2 better than the JH1 domain suggesting that there are at least two sites of interaction for SSG1 in JAK2. SSG1 contains three SRPX5 (sushi-repeat-containing protein, X-linked, domain 5) domains. All three of these SRPX5 domains interact preferentially with the kinase-active form of JAK2. Our results suggest that the SRPX5 domain is a novel protein-protein interaction domain, most likely a phosphotyrosine binding domain. To determine the physiological consequences of the interaction between SSG1 and JAK2, we show that SSG1 does not alter JAK2 kinase activity and only modestly increases GH-dependent phosphorylation of Stat5b on Tyr694. In contrast, SSG1 enhances phosphorylation of Stat3 on Tyr705 in the presence and absence of GH. Phosphatase experiments suggest that SSG1 enhances GH-independent phosphorylation of a different, currently unidentified amino acid in Stat3, and negatively regulates the GH-responsive c-fos promoter construct. SSG1 co-localizes with markers for both the endoplasmic reticulum and Golgi. By confocal microscopy, I show that a portion of JAK2 co-localizes with SSG1 in these subcellular compartments. SSG1, which contains a signal peptide, is a secreted protein. Secretion is blocked by Brefeldin A. JAK2 enhances both the secretion and cleavage of SSG1, assessed by TCA precipitation of medium and immunofluorescence. Together, my results show that SSG1 interacts with JAK2 and regulates downstream signaling of JAK2. JAK2 enhances the secretion and potentially extracellular cleavage of SSG1.