ABSTRACT
Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.
Identification of phosphorylation sites and binding pockets for modulation of Na
V
1.5 channel by Fyn tyrosine kinase
