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

Abstract

Janus tyrosine kinase 3 (Jak3) is essential for signaling by interleukin-2 (IL-2) family cytokines and proper immune function. Dysfunctional regulation of Jak3 may result in certain disease states. However, the molecular mechanisms governing Jak3 activation are not fully understood. In this study, we used a functionalproteomics approach to identify two novel tyrosine phosphorylation sites within Jak3, Y904 and Y939, which are conserved among Jak family proteins. By using phosphospecific antibodies, both residues were observed to be rapidly induced by stimulation of cells with IL-2 or other ␥c cytokines. Mechanistic studies indicated that Y904 and Y939 regulate Jak3 activities. A phenylalanine substitution at either site greatly reduced Jak3 kinase activity in vitro and its ability to phosphorylate signal transducer and activator of transcription 5 (Stat5) in vivo, suggesting that phosphorylation of these previously unrecognized residues positively regulates Jak3 activity. Y904 and Y939 were required for optimal ATP usage by Jak3, while phosphorylation of Y939 preferentially promoted Stat5 activity in intact cells. Together, these findings demonstrate positive functional roles for two novel Jak3 phosphoregulatory sites which may be similarly important for other Jak family members. Identification of these sites also provides new therapeutic opportunities to modulate Jak3 function.

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

“…Immunoprecipitated Jak3 was washed with kinase buffer (50 mM Hepes-NaOH (pH 7.4), 10 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM DTT, 20 g/ml aprotinin, 10 g/ml leupeptin, 1 g/ml pepstatin A) and incubated with 200 M ATP and purified protein kinase A catalytic subunit (PKAc) (Invitrogen) as indicated in the figure legends. Kinase reactions were carried out at 32°C for 30 min followed by vigorous washing of the beads with cold kinase wash buffer as described previously (31). For [␥-32 P]ATP radiolabeled kinase assays using recombinant Jak3, Hek293 cells were transfected with wild type (WT) Jak3 or kinase-dead Jak3 K855A (31) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).…”

“…Kinase reactions were carried out as described previously (31) at 30°C for 20 min. For PKA kinase assays, untreated MT-2 cells were lysed, and Jak3 was immunoprecipitated and bound to PAS beads as described previously (31). Immunoprecipitated Jak3 was washed with kinase buffer (50 mM Hepes-NaOH (pH 7.4), 10 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM DTT, 20 g/ml aprotinin, 10 g/ml leupeptin, 1 g/ml pepstatin A) and incubated with 200 M ATP and purified protein kinase A catalytic subunit (PKAc) (Invitrogen) as indicated in the figure legends.…”

“…3A, graph). Phosphorylation of Tyr 980 , Tyr 981 , and Tyr 939 have been shown to regulate Jak3 enzymatic activity (31,42). Given the importance of Tyr 939 for Stat5 association, we examined Fsk treatment on Jak3 Tyr 939 phosphorylation (Fig.…”

Interleukin-2 Receptor β Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules

“…Solubilization of Proteins, Immunoprecipitation, Western Blot, and Mass Spectrometry Analysis-Cells were pelleted, lysed, and subjected to immunoprecipitation and Western blot analysis as reported previously (31). For total cell lysate, protein concentration was determined by the bicinchoninic acid method (Pierce).…”

“…The pcDNA3.1 human JAK3 and STAT5B cDNAs (OriGene) were obtained as described previously (31,34). Mutant forms of IL-2R␤ were prepared using the QuikChange site-directed mutagenesis kit (Stratagene) according to the instructions of the manufacturer.…”