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Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

Abstract

To drive lymphocyte proliferation and differentiation, common γ-chain (γc) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γc-receptor complexes, but how their respective catalytic activities contribute to signaling over time is not known. Here we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring phosphorylation of the transcription factor STAT5 over 20 h in CD4+ T cells stimulated with interleukin 2 (IL-2), we document a second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and entry to S phase. An inhibitor-resistant JAK3 mutant (C905S) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2–driven T cell proliferation and will find broad utility in studies of γc-receptor signaling.

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Figure 1: Chemical structure of JAK3i.
Figure 2: Early and late signaling events are differentially affected by JAK3i.
Figure 3: C905S JAK3 rescues JAK3i effects in primary CD4+ T cells.
Figure 4: A second wave of STAT5 signaling is highly sensitive to JAK3 inhibition.
Figure 5: Sustained JAK3 activity is required for S phase entry.
Figure 6: JAK3 inhibition blocks IL-2–driven proliferation in vivo.

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Acknowledgements

We thank B. Au-Yeung, J. Carelli, H. Wang and K. Shokat for helpful discussion and suggestions and A. Roque for animal husbandry. This work was funded by the Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center (A.W.) and the Howard Hughes Medical Institute (A.W. and J.T.). G.A.S. was supported by the University of California, San Francisco Medical Scientist Training Program and the US National Institute of Allergy and Infectious Diseases (F30AI120517-01).

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G.A.S. designed experiments, performed chemical synthesis and biological experiments and wrote the manuscript. K.U. performed chemical synthesis. A.W. designed experiments and wrote the manuscript. J.T. designed experiments and wrote the manuscript.

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Correspondence to Jack Taunton.

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Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–12. (PDF 3570 kb)

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Synthetic Procedures (PDF 151 kb)

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Smith, G., Uchida, K., Weiss, A. et al. Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling. Nat Chem Biol 12, 373–379 (2016). https://doi.org/10.1038/nchembio.2056

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