Activation of Jak2 Catalytic Activity Requires Phosphorylation of Y¹⁰⁰⁷ in the Kinase Activation Loop

Abstract

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y¹⁰⁰⁷ and Y¹⁰⁰⁸, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y¹⁰⁰⁷ and Y¹⁰⁰⁸ are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y¹⁰⁰⁷, or both Y¹⁰⁰⁷ and Y¹⁰⁰⁸, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y¹⁰⁰⁸ to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in γ2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y¹⁰⁰⁷ to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K⁸⁸²E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y¹⁰⁰⁷ in Jak2 regulation and function.