Characterization of the exokinase activities of specific activity states of recombinant rat Janus kinase 2

Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase crucial to signal propagation in the signal transduction pathways of multiple cytokines. The activity of JAK2 activation is tightly controlled by multiple regulatory factors such as the phosphorylation status of the activation loop tyrosines and the interaction of pseudokinase domain with the kinase domain. JAK2 exists in at least two activity states, a basally active JAK2 prior to the activation loop tyrosine phosphorylation and a fully active JAK2 following the phosphorylation of tyrosines 1007 and 1008 residues in the activation loop. A gain-of-function mutation (V617F) in the pseudokinase domain of JAK2 is frequently present in patients with myeloproliferative disorders (MPDs). The exact mechanism of JAK2(V617F) hyperactivation is not known but it is generally thought that the V617F mutation results in the loss of autoinhibition exerted by the pseudokinase domain on the kinase domain of JAK2 resulting in a marked increase in JAK2's catalytic activity. We have shown that V617F mutation alone is not sufficient for the hyperactivation of the JAK2(V617F) mutant, and that the mutant will still require phosphorylation of tyrosines 1007 and 1008 in the activation loop. These observations suggest that the phosphorylation of activation loop tyrosines is the dominant activation process which takes precedence over relief of autoinhibition, and therefore, the phosphorylation of activation loop tyrosines is indispensable for the maximal activation of JAK2 variants, including the JAK2(V617F) mutant.The lack of suitable substrates has been the primary limitation in the development of JAK exogenous assays. We evaluated the cytoplasmic domains of the murine erythropoietin receptor (mEpoR), human prolactin receptor (hPRLR) and the human growth hormone receptor (hGHR) as possible substrates for the development of JAK2-mediated exogenous assay. The availability of a reliable assay will greatly enhance our understanding of the affect of regulatory factors on JAK2 structural conformation and its catalytic activity states. With the delineation of the steps involved in JAK2 activation and regulation under both normal and pathological conditions, it is possible to develop a selective JAK2 inhibitor that targets a distinguishing feature in the de-regulated JAK2 that is absent in normal JAK2.