Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.

Autor: Korniski B; Pfizer Inc, Global Research and Development, St Louis Laboratories, 700 Chesterfield Parkway West, St Louis, MO 63017-1732, USA., Wittwer AJ, Emmons TL, Hall T, Brown S, Wrightstone AD, Hirsch JL, Gormley JA, Weinberg RA, Leone JW, Day JE, Chrencik JE, Sommers CD, Fischer HD, Tomasselli AG
Jazyk: angličtina
Zdroj: Biochemical and biophysical research communications [Biochem Biophys Res Commun] 2010 May 28; Vol. 396 (2), pp. 543-8. Date of Electronic Publication: 2010 May 08.
DOI: 10.1016/j.bbrc.2010.04.141
Abstrakt: The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.
(Copyright (c) 2010 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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