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

The use of a selective JAK3 covalent inhibitor reveals two distinct temporal waves of STAT5 phosphorylation. The inhibitor more potently targets the second wave, which is required for cell cycle progression and T cell proliferation. To drive lymphocyte proliferation and differentiation, common γ-chain (γ c ) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γ c -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 γ c -receptor signaling.