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

The Janus kinase (Jak) family of cytoplasmic tyrosine kinases associates with a variety of cell surface receptors to perform essential roles for transducing intracellular signals (9, 15). There are four Jak family members in vertebrates: Jak1, Jak2, Jak3, and Tyk2. While Jak1, Jak2, and Tyk2 are ubiquitously expressed, Jak3 is predominantly expressed in hematopoietic cells (20, 30, 41). Jak3 specifically associates with the cytokine receptor γ common (γc) chain and can be activated by interleukin-2 (IL-2) family cytokines such as IL-2, IL-4, IL-7, and IL-9 (40, 45). Inhibitory mutations in Jak3 or its binding partner, γc, can result in severe combined immunodeficiency (SCID) syndrome in humans and mice, which is clinically manifested by limited numbers of T, natural killer, and functional B cells (34, 35). Hyperactivation of Jak3 has also been associated with diseases such as asthma (31) and cancers of the immune system (44). The restricted expression and function of Jak3 has made it a promising target for controlling these diseases (6, 33, 39). The activation of Jak proteins contributes to multiple cellular processes, including cell growth, proliferation, and differentiation (1). Following receptor engagement by cytokines, the activation of Jak proteins is believed to occur by auto- or transphosphorylation of key tyrosine residues located within their activation loops (12). Stimulation of hematopoietic cells with IL-2 family growth factors results in the phosphorylation and enzymatic activation of γc-associated Jak3 and another Jak family member, Jak1, which may bind to a cytokine-specific receptor subunit cooperatively with γc (19). Activated Jak1 and/or Jak3 then phosphorylate tyrosine residues on the associated receptors to produce docking sites for SH2- or PTB-containing proteins such as signal transducer and activator of transcription 5 (Stat5) (14, 24, 25), leading to their phosphorylation and subsequent activation. These proteins then regulate many downstream events, including gene transcription. Phosphorylation plays a critical role in regulating Jak3 kinase activity. It has been reported that two adjacent tyrosines located in the Jak3 kinase activation loop are phosphorylated to positively (Y980) or negatively (Y981) regulate its catalytic activity (47). 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-β, 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). Suppressor of cytokine signaling family proteins form a classical negative feedback loop to attenuate cytokine signaling that can also act through the Jak/Stat pathway (2). To determine whether other phosphosites exist, we mutated the three known residues, Y980, Y981, and Y785, and found no significant change in total tyrosine phosphorylation. Using mass spectrometry, we identified two additional phosphotyrosines in Jak3 at Y904 and Y939. Phosphospecific antibodies confirmed that phosphorylation of Jak3 on these sites occurred in response to IL-2 and other IL-2 family cytokines in multiple cell types, including primary human T cells. Phenylalanine substitution of these residues inhibited Jak3 tyrosine phosphorylation and catalytic activity. Evidence is provided to suggest that Y904 is required for ATP binding while Y939 may be required for substrate association. More importantly, these sites are conserved in other Jak family members, suggesting a universal regulatory role for these tyrosine residues.