Jak3 is a key immunoregulatory enzyme responsible for promoting normal and abnormal immune responses. Thus, understanding its mechanism of regulation is fundamentally important. For this work, two novel tyrosine phosphorylation sites in Jak3 were identified by mass spectrometry and further characterized with site-specific phospho-specific antibodies. Y904 and Y939 were determined to be rapidly and transiently phosphorylated in response to IL-2 in YT, Kit225, and primary human T cells. IL-9 similarly activated the phosphorylation of Jak3 at Y904 and Y939 in Kit225 cells. The tyrosine phosphorylation kinetics of Y904 or Y939 peaked at 2 min and returned to baseline by 60 min in YT and human T lymphocytes. In contrast, Kit225 cells showed protracted phosphorylation kinetics, with pY904 and pY939 only returning to basal levels after 120 min of IL-2 stimulation. Additionally, Y904 and Y939 were readily autophosphorylated and required for Jak3 catalytic activity to phosphorylate a defined substrate. This is likely due to the requirements for Y904 and Y939 in ATP and substrate binding.
For protein kinases, phosphorylation of key residues within the activation loop, which is localized between subdomains VII and VIII of the kinase domain, induces a conformational change which facilitates the access of substrates to the active site. Hence, phosphorylation of these residues often increases the intrinsic catalytic activities of kinases (29
), as is true for Y1162 in the insulin receptor kinase (13
). For Jak family proteins, two adjacent tyrosine residues within the activation loop have been implicated in the control of their catalytic activities. In Jak3, Y980 is a positive regulatory site while Y981 negatively controls its activity (47
). Similarly, mutation of positionally conserved Y1007 to phenylalanine in Jak2 blocked its activation while phenylalanine substitution of Y1008 had no effect (7
Jak proteins have seven regions of sequence similarity named Janus homology (JH) domains. The tyrosine kinase domain is localized to the C terminus in JH1. Jak proteins are unique in having a catalytically inactive pseudokinase domain (JH2), which has been shown to regulate their kinase activity (36
). The N-terminal JH4 to JH7 domains of Jak proteins are involved in receptor association. This region has a band 4.1, ezrin, radixin, and moesin (FERM) domain (10
). Several mutations in the FERM domain of Jak3 were found in SCID patients (9
). Except for tyrosine phosphorylation within the activation loop of the kinase domain, few studies have expanded the functional roles of other putative phosphorylation sites in Jak proteins. Among the four Jak kinases, autophosphorylation of Jak2 may be the best characterized. Indeed, Y221 in the FERM domain and Y570 in the JH2 domain are sites of autophosphorylation in Jak2 that differently regulate its catalytic activity (3
). Phosphorylation of Jak2 on Y119 in the FERM domain was found to down-regulate erythropoietin signaling by promoting the dissociation of Jak2 from the receptor complex (8
). Y813 in the JH2 domain of Jak2 represents another site of phosphorylation which is required for binding to the adaptor protein SH2-Bβ, 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-Bβ (23
). Phosphorylation of Jak2 on S523 was recently demonstrated to function as a regulatory feedback mechanism to dampen the activity of this enzyme (28
). While little is known about serine phosphorylation in other Jak proteins, we also observed that Jak3 was serine phosphorylated upon IL-2 stimulation (data not shown). Additional work seeks to identify this serine residue(s).
Extending the current model, our data indicate that phosphorylation of both Y904 and Y939 positively regulates Jak3 activity. This is based on our observations that mutation of either site to phenylalanine impairs the autocatalytic activity of Jak3, as well as its ability to phosphorylate substrates such as GST-γc in vitro (Fig. ). Phenylalanine mutation of either Y904 or Y939 impaired its ability to stimulate Stat5 tyrosine phosphorylation and transcriptional activity (Fig. ). Our data provide insight into the molecular mechanisms by which phosphorylation of Y904 and Y939 positively regulates Jak3 activity. Based upon the crystal structure of the Jak3 kinase domain, Y904 is likely confined to the ATP binding pocket between two amino acids shown to make contact with the ATP analogue ANF941(Fig. ). Indeed, phenylalanine substitution of Y904 increased the Km of Jak3 for ATP from 1.31 to 4.52 μM and reduced the Vmax of Jak3 toward GST-γc by approximately 50% (Fig. ). Although Y939 lies outside the proposed ATP binding domain, this residue also changed the Km of Jak3 for ATP from 1.31 to 1.79 μM and reduced the Vmax of Jak3 toward GST-γc by approximately 50%. Nonetheless, these data indicate that Y904 and Y939 are important for Jak3 ATP binding affinity and that phosphorylation of these sites promotes Jak3 catalytic activity.
Interestingly, Y939 is localized to the α-helix E of the C lobe, suggesting that phosphorylation of this site may influence substrate interaction. Moreover, phosphorylation of this residue is predicted to create a Stat5 SH2 binding site (pY[VLTFIC]XX). To test this notion, phenylalanine substitution of Y939 in Jak3 was found to result in reduced catalytic activity in vitro (Fig. ) and completely blocked its ability to activate Stat5a in vivo (Fig. ). Moreover, a 10-mer peptide harboring pY939, but not its nonphosphorylated counterpart, was able to capture endogenous Stat5 from YT-cell lysates (Fig. ). It is also noteworthy that this peptide readily coassociated with other, unknown, tyrosine-phosphorylated proteins, indicating that phosphorylated Y939 may regulate other signaling molecules. These data lead us to propose that Y939 serves two roles in the Jak3 activation mechanism. When phosphorylated, it may enhance access to the catalytic cleft for substrates lacking an SH2 domain (e.g., GST-γc) and may also provide a docking site for SH2 domain-containing proteins such as Stat5. Results in Fig. 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
). 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. This may be especially important in tumor models where constitutively active Jak3 may activate substrates independently of a receptor.
In summary, we have identified Y904 and Y939 in Jak3 as two novel sites of cytokine-mediated phosphorylation. Phosphoantibodies that specifically recognized these residues confirmed that Jak3 Y904 and Y939 are rapidly and transiently induced in YT, Kit225, and primary human T cells. Y904 and Y939 positively regulate the enzymatic activity of Jak3 and its substrate Stat5. Lastly, we provide evidence that phosphorylation of Y904 and Y939 of Jak3 regulates Stat5 and ATP binding activity. These data provide new insight into the mechanisms that regulate Jak3 activation and its downstream signaling.