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 1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y 1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in ␥2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y 1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K 882 E) 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 Y 1007 in Jak2 regulation and function.The Janus tyrosine kinase (Jak) family consists of four members, Jak1, Jak2, Jak3, and Tyk2. Over the past several years, it has become evident that these kinases play a critical role in the functions of receptors of the cytokine receptor superfamily (4,7,8,15,19). In general, the Jaks associate with the membraneproximal region of one or more of the receptor chains constitutively or following ligand-induced receptor aggregation. Following ligand binding, there is a rapid induction of tyrosine phosphorylation of the receptor-associated Jaks and a striking increase in their catalytic activity in in vitro kinase assays of the immunoprecipitated Jaks. It is hypothesized that ligand-induced receptor aggregation results in the aggregation of the associated Jaks, which allows Jak auto-or transphosphorylation at sites that regulate catalytic activity. The activated Jaks subsequently tyrosine phosphorylate the receptor chains as well as a variety of substrates that are recruited to the activated receptor complex.The role of tyrosine phosphorylation of Jaks in regulation of their intrinsic kinase activity, although hypothesized, has only recently been addressed. The regulation of tyrosine kinase activity has been shown to occur in two distinct manners. The family of Src kinases are regulated through the tyrosine phosphorylation of a carboxyl-terminal site (2, 14, 16). It is speculated that this site interacts with the SH2 domain to induce an inactive conformation of the kinase domain. Activation of kinase activity is therefore associated with dephosphorylation of the carboxyl-terminal site. Alternatively, a number of receptor tyrosine kinases are catalytically activated by tyrosine ph...

Signalling cross-talk between hepatocyte nuclear factor 4α and growth-hormone-activated STAT5b

“…This inhibition was observed when using both rat and human HNF4α. The inhibitory effect of HNF4α was manifested at the level of GH-stimulated JAK2 phosphorylation of Tyr 1007/1008 , which is causally linked to the activation of JAK2 catalytic activity [38]. By contrast, HNF4α did not inhibit interferon-γ -stimulated STAT1 transcriptional activity, which is mediated by the interferon-γ receptor and requires both JAK1 and JAK2 [39,40].…”

Activation of Stat5 by platelet-derived growth factor (PDGF) is dependent on phosphorylation sites in PDGF β-receptor juxtamembrane and kinase insert domains

“…We also estimated the kinase activity of JAK1 in response to PDGF-BB. For that purpose, PAE cells expressing PDGF-b-receptor, were stimulated with PDGF-BB, subjected to immunoprecipitation with antiserum against JAK1 and assayed for the kinase activity of JAK1 with an exogenous peptide corresponding to the JAK2 phosphorylation site (Feng et al, 1997;Silvennoinen et al, 1993a). As shown in Figure 9c, stimulation with PDGF-BB induced the kinase activity of JAK1 as compared to non stimulated cells.…”

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

“…We created expression vectors for an enhanced green fluorescent protein chimera in which the fluorescent protein was fused to the amino-terminus of rat JAK2 (EGFP/rJAK2), along with a kinase-inactive control protein (EGFP/ rJAK2(K882E)) [27,28]. Following transient transfection, all three fluorescent chimeric proteins were produced in COS-7 cells and the characteristic green fluorescence was observed microscopically for all recombinant proteins (Figure 1).…”