Identification of SH2-Bβ as a Substrate of the Tyrosine Kinase JAK2 Involved in Growth Hormone Signaling

Abstract

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bβ, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bβ (SH2-Bβc), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bβ or SH2-Bβc and murine JAK2, both SH2-Bβc and SH2-Bβ coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bβc also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bβ with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bβc or GST-SH2-Bβ fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bβ. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bβ in 3T3-F442A cells, further suggesting the involvement of SH2-Bβ in GH signaling. Consistent with SH2-Bβ being a substrate of JAK2, SH2-Bβc is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bβ was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bβ and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.