| The kinase Jak2 is a member of the Janus family of non-receptor tyrosine kinases. One major impediment to understanding the role that Jak2 plays in physiology and pathophysiology is the lack of a specific Jak2 inhibitor. We used several strategies to circumvent this problem. First, using Jak2 -/- cells, we examined the role of Jak2 in regulating angiotensin II-dependent ERK2 activity. We found that, contrary to previously published work, Jak2 is required for inactivation of ERK2 after angiotensin II treatment. In response to angiotensin II, Jak2 induces expression of MAP kinase phosphatase-1 (MKP-1), a protein that dephosphorylates and inactivates ERK2.; Second, using stable expression of a Jak2 dominant negative mutant that specifically suppresses endogenous Jak2 kinase activity, we found that Jak2 mediates oxidative stress-induced apoptosis of vascular smooth muscle cells. In response to hydrogen peroxide treatment, Jak2 induces expression of the pro-apoptotic Bax protein. This causes a loss of mitochondrial transmembrane potential, cleavage of Caspase-9, and subsequent apoptosis.; Third, we attempted to identify novel Jak2 inhibitors. For this, we used homology modeling to analyze the structure of the Jak2 kinase domain, and we identified a previously unknown amino acid interaction that is required for activation of Jak2. This interaction, consisting of two distinct hydrogen bonds between Jak2 residues Glu 1024 and Arg 1113, may be a suitable target for drug design aimed at disabling Jak2 function. Additionally, we used high-throughput compound docking in silico to identify a novel Jak2 inhibitor. This compound, cyclohexane-1,2,3,4,5,6-hexabromo- (designated Compound 7) potently inhibits Jak2 autophosphorylation in a time- and dose-dependent manner.; In conclusion, using Jak2 -/- cells, stable expression of a Jak2 dominant negative mutant, and structure-function studies, we successfully circumvented the problems that lack of a Jak2-specific inhibitor pose. In doing so, we identified novel roles for Jak2 kinase function in regulation of angiotensin II-dependent ERK2 signaling and in oxidative stress-induced apoptosis of vascular smooth muscle cells. Additionally, we improved our understanding of Jak2 structure by identifying a previously unknown amino acid interaction within the Jak2 kinase domain that is required for Jak2 kinase function, and identified a novel Jak2 inhibitor. |
