title: Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.
abstract: 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.
authors.affiliation: Pfizer Inc, Global Research and Development, St Louis Laboratories, 700 Chesterfield Parkway West, St Louis, MO 63017-1732, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Korniski
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Wittwer
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Emmons
authors.forename: Troii
authors.initials: T
authors.lastname: Hall
authors.forename: Stacy
authors.initials: S
authors.lastname: Brown
authors.forename: Ann D
authors.initials: AD
authors.lastname: Wrightstone
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Hirsch
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Gormley
authors.forename: Robin A
authors.initials: RA
authors.lastname: Weinberg
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Leone
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Day
authors.forename: Jill E
authors.initials: JE
authors.lastname: Chrencik
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Sommers
authors.forename: H David
authors.initials: HD
authors.lastname: Fischer
authors.forename: Alfredo G
authors.initials: AG
authors.lastname: Tomasselli
mesh_terms: D000818:Animals; D002384:Catalysis; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification*; D053616:Janus Kinase 3 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification*; D051379:Mice; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D053634:TYK2 Kinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification*
chemical_list: D053614:Janus Kinase 2; D053616:Janus Kinase 3; D053634:TYK2 Kinase