Insights into kinetic mechanism of Janus kinase 3 and its inhibition by tofacitinib.
JAK3 kinase plays a critical role in several cytokine signaling pathways involved in immune cell development and function. The studies presented in this report were undertaken to elucidate the kinetic mechanism of the JAK3 kinase domain, investigate the role of activation loop phosphorylation in regulating its catalytic activity, and examine its inhibition by the anti-rheumatoid arthritis drug, tofacitinib. Phosphorylation of two Tyr residues in JAK3's activation loop has been reported to impact its kinase activity. The recombinant JAK3 kinase domain used in our studies was heterogeneous in its activation loop phosphorylation, with the non-phosphorylated protein being the dominant species. Kinetic analysis revealed similar kinetic parameters for the heterogeneously phosphorylated JAK3, JAK3 mono-phosphorylated on Tyr 980, and the activation loop mutant YY980/981FF. Bisubstrate and product inhibition kinetic results were consistent with both sequential random and sequential ordered kinetic mechanisms. Solvent viscosometric experiments showed perturbation of kcat, suggesting the phosphoryl transfer step is not likely rate limiting. This was supported by results from quench-flow experiments, where a rapid burst of product formation was observed. Kinetic analysis of JAK3 inhibition by tofacitinib indicated inhibition is time dependent, characterized by on- and off-rate constants of 1.4 ± 0.1 μM-1s-1 and 0.0016 ± 0.0005 s-1, respectively.
['Hekmatnejad|Mohammad|M|', 'Conwell|Sara|S|', 'Lok|Stephen M|SM|', 'Kutach|Alan|A|', 'Shaw|David|D|', 'Fang|Eric|E|', 'Swinney|David C|DC|']
D000251:Adenosine Triphosphatases D000818:Animals D002384:Catalysis D004305:Dose-Response Relationship, Drug D006801:Humans D020128:Inhibitory Concentration 50 D007313:Insecta D053616:Janus Kinase 3 D007700:Kinetics D009154:Mutation D010766:Phosphorylation D010880:Piperidines D047428:Protein Kinase Inhibitors D011743:Pyrimidines D011758:Pyrroles D061987:Sf9 Cells D015398:Signal Transduction D012997:Solvents D014783:Viscosity
Kinetic mechanism Tofacitinib Sequential random JAK3 activation mechanism Time dependent inhibition