Tyrosine 813 Is a Site of JAK2 Autophosphorylation Critical for Activation of JAK2 by SH2-Bβ

Abstract

The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B␤ to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B␤ to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B␤-binding site within JAK2 that is required for SH2-B␤ to enhance activation of JAK2.The Janus kinase family of tyrosine kinases (JAK1, JAK2, JAK3, and Tyk2) plays an essential role in the signaling by all members of the cytokine receptor superfamily. The JAKs promote growth, proliferation, and/or differentiation of many cell types (1, 15). Activation of the JAKs occurs upon ligand binding to its receptor. Activation is thought to occur as a consequence of two JAK molecules being brought into close enough proximity to allow for rapid trans-phosphorylation of the activation loop of each kinase (15). The activated JAK molecules then phosphorylate multiple targets, including the JAKs themselves, the associated receptors, and multiple signaling molecules such as the signal transducers and activators of transcription (STATs) (4,10,16,17). Dysregulation of JAKs can lead to a host of physiological problems, including diseases of the immune system (6) and cancer (22,29,40).Among the JAKs, JAK2 is activated by more than two-thirds of the known cytokine receptor ligands, including growth hormone (GH), prolactin, erythropoietin, and leptin, making it the most studied of the JAK family members (14). Autophosphorylation of JAK2 is an important step in regulating signaling as it leads to activation of the kinase. Autophosphorylation also results in the production of potential docking sites for downstream signaling molecules containing phosphotyrosine binding Src homology 2 (SH2) domains, such as the adapter protein, SH2-B, and the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS-1). Identification of the autophosphorylation sites on JAK2, therefore, is critical for advancing our understanding of how these kinases signal, and could provide potential targets for pharmaceutical ...

Phosphorylation of Human Jak3 at Tyrosines 904 and 939 Positively Regulates Its Activity

“…5 indicate that Y904 is less effective than Y939 in mediating Stat5 activation. These data may explain how Stat5 can be activated by Jak2 or Jak3 independently of a receptor involvement (23,47). Based on our findings, we hypothesize that, in the absence of cytokine receptors, phosphorylation of Jak3 at Y939, and possibly Y904, is required for Stat5 association and subsequent activation.…”

“…Three tyrosine residues in Jak3, Y980, Y981, and Y785, were previously identified as phosphoregulatory sites (23,47). Mutational analysis was used to reexamine the regulatory role of these tyrosines and to determine whether Jak3 VOL.…”

“…Y813 in the JH2 domain of Jak2 represents another site of phosphorylation which is required for binding to the adaptor protein SH2-B␤, which further enhances the activity of this enzyme. Positionally conserved corresponding tyrosine 785 in Jak3 is phosphorylated in response to IL-2 and is also important for binding to SH2-B␤ (23). Phosphorylation of Jak2 on S523 was recently demonstrated to function as a regulatory feedback mechanism to dampen the activity of this enzyme (28).…”

“…Phosphorylation of Jak proteins can also provide binding sites for other signaling molecules. For example, phosphorylation of Jak3 on Y785 has been reported to create a binding site for the adaptor protein SH2B-␤, although the functional significance of this interaction is unknown (23). Negative regulatory mechanisms of Jak3 activity include dephosphorylation by CD45 and T-cell protein tyrosine phosphatase (17,38).…”

Aberrant signal transduction pathways in myeloproliferative neoplasms

“…Some of these tyrosine residues exert positive (Y221) and other negative (Y119, Y570) effects on signaling by JAK2. [111][112][113] Y813 is a recruitment site for SH2-containing proteins, 114 such as SH2B, which can promote homodimerization of JAK2. 115 In theory, the constitutive activation of JAK2 V617F might promote a different pattern of phosphorylated tyrosines from that of wild-type JAK2.…”