title: Differential kinetics and inhibition of purified recombinant tyrosine kinase 2 (TYK-2) and its catalytic domain JH-1.
abstract: The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.
authors.affiliation: Pfizer Global Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway West, St. Louis, MO 63017, USA. Thomas.l.emmons@pfizer.com
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Emmons
authors.forename: Ann D
authors.initials: AD
authors.lastname: Wrightstone
authors.forename: Eric T
authors.initials: ET
authors.lastname: Baima
authors.forename: Stacy
authors.initials: S
authors.lastname: Brown
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Sommers
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Hirsch
authors.forename: Lyle E
authors.initials: LE
authors.lastname: Pegg
authors.forename: Robin A
authors.initials: RA
authors.lastname: Weinberg
authors.forename: H David
authors.initials: HD
authors.lastname: Fischer
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Wittwer
authors.forename: Alfredo G
authors.initials: AG
authors.lastname: Tomasselli
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053634:TYK2 Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D053634:TYK2 Kinase; C070139:TYK2 protein, human