Identification of SH2-B beta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling

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 homolog 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-B beta, as a JAK2-interacting protein, The carboxyl terminus of SH2-B beta (SH2-B beta c), 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-B beta or SH2-B beta c and murine JAK2, both SH2-B beta c and SH2-B beta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-B beta c 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-B beta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-B beta c or GST-SH2-B beta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-B beta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-B beta in 3T3-F442A cells, further suggesting the involvement of SH2-B beta in GH signaling. Consistent with SH2-B beta being a substrate of JAK2, SH2-B beta c is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells, SH2-B beta 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-B beta 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.