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

To drive lymphocyte proliferation and differentiation, common γ-chain (γ(c)) cytokine receptors require hours to days of sustained stimulation. While JAK1 and JAK3 kinases are found together in all γ(c)-receptor complexes, it is not known how their respective catalytic activities contribute to signaling over time. Here, we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring STAT5 phosphorylation over 20 hours in IL-2-stimulated CD4+ T cells, we document a previously unappreciated 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 S-phase entry. An inhibitor-resistant JAK3 mutant (Cys905Ser) 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.