title: Identification of SH2-Bbeta 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-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), 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-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac 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-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta 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-Bbeta 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.
authors.affiliation: Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
authors.forename: L
authors.initials: L
authors.lastname: Rui
authors.forename: L S
authors.initials: LS
authors.lastname: Mathews
authors.forename: K
authors.initials: K
authors.lastname: Hotta
authors.forename: T A
authors.initials: TA
authors.lastname: Gustafson
authors.forename: C
authors.initials: C
authors.lastname: Carter-Su
mesh_terms: D017667:Adipocytes; D017398:Alternative Splicing; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D018121:Receptors, Cytokine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D014018:Tissue Distribution; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D014443:Tyrosine; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; C507927:Jak2 protein, rat; D053614:Janus Kinase 2