Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

Abstract

Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr 221 and Tyr 570 as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr 221 , Tyr 570 lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2 Y570F ) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr 570 does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr 570 in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr 570 -dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.Type I cytokines mediate a plethora of physiologic processes, ranging from hematopoietic and immune functions (such as those mediated by erythropoietin [EPO] and the interleukins [ILs]) to growth and neuroendocrine responses (such as those mediated by growth hormone and leptin) (12,14,16,23). These actions are mediated by the activation of cytokine receptor proteins found on the surface of target cells. Cytokine receptors each contain an extracellular domain that recognizes its specific cytokine ligand, a single transmembrane domain, and an intracellular domain that, although devoid of enzymatic activity, transmits intracellular signals by means of an associated Jak family tyrosine kinase. Ligand binding activates the associated intracellular Jak kinase, resulting in the tyrosine phosphorylation of the Jak kinase and the intracellular domain of the cytokine receptor. These tyrosine phosphorylation events mediate the recruitment of downstream signaling molecules that contain phosphotyrosine-binding SH2 domains (such as STAT proteins) (12, 16); tyrosine phosphorylation may also mediate other regulatory events during cytokine signaling (8, 31).The Jak kinase family contains four members: Jak1 to Jak3 and Tyk2 (12, 16). Of these, Jak1, Jak2, and Tyk2 are ubiquitously expressed, while Jak3 is found predominantly in immune and hematopoietic tissues. Jak kinases are composed of four conserved domains. The NH 2 -terminal FERM domain is required for interaction with cytokine receptors (24, 30), while the adjacent SH2-like fold has no known function. The COOH-terminal portion of Jak kinases contains a kinase-like JH2 domain that is devoid of enzymatic activity but that inhibits the activity of the COOH-terminal JH1 tyrosine kinase domain (10,19,22,28,29).Our laboratory studies s...

JAK1 and Tyk2 Activation by the Homologous Polycythemia Vera JAK2 V617F Mutation

“…These results strongly suggest that one role of the JH2 domain, at least in JAK2, is to keep the kinase domain inactive in the absence of cytokines. Furthermore, upon phosphorylation, Tyr 570 in the JH2 domain of JAK2 negatively regulates activity (12,13). This is in contrast to other phosphorylation sites, such as the positively acting Tyr 221 (13), Tyr 813 (14) and, of course, the activation loop Tyr 1007 , which is required for activation (15).…”

Mutation Profile of JAK2 Transcripts in Patients with Chronic Myeloproliferative Neoplasias

“…The residue adjacent to G571, tyrosine 570 (Y570), is one of the most important tyrosine phosphorylation sites (of all 49 tyrosine residues) in JAK2; autophosphorylation of Y570 plays an important role in the mechanism that downregulates JAK2 kinase activity. 11 Thus, the G571 mutation may cause a conformational change such that the adjacent Y570 is no longer accessible and phosphorylated, resulting in a constitutively active kinase.…”

Hematopoietic cytokine receptor signaling

“…A more detailed analysis of the Jak2 JH2 region using recombinant proteins indicates that this domain suppresses basal JAK2 activity by lowering the V max of the kinase without affecting its K m for a substrate peptide (Saharinen et al, 2003). Additional studies have revealed the presence of three inhibitory regions (1-3) that regulate the autoinhibitory activity of this kinase as well as a novel phosphorylation site at tyrosine 570 that regulates responses to cytokines (Saharinen et al, 2003;Feener et al, 2004). Taken together, these studies indicate that the JH2 domain is critical in the ability of JAKs to regulate themselves as well as mediate cytokine-induced responses.…”

“…Of these, TYK2 was the first to be discovered (Firmbach-Kraft et al, 1990) and bore some resemblance to known tyrosine kinases. Shortly thereafter, cDNAs encoding JAK1 (Wilks et al, 1991), JAK2 (Harpur et al, 1992) and JAK3 Rane and Reddy, 1994;Takahashi and Shirasawa, 1994) were isolated using various cloning strategies.…”