Differential Kinetics and Inhibition of Purified Recombinant Tyrosine Kinase 2 (TYK-2) and Its Catalytic Domain JH-1

Author(s): Thomas L. Emmons, Ann D. Wrightstone, Eric T. Baima, Stacy Brown, Cynthia D. Sommers, Jeffrey L. Hirsch, Lyle E. Pegg, Robin A. Weinberg, H. David Fischer, Arthur J. Wittwer, Alfredo G. Tomasselli

Journal Name: Protein & Peptide Letters

Volume 19 , Issue 5 , 2012

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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.

Keywords: Activation, autophosphorylation, IC50, inhibition, CP-690550, PF-956980

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Article Details

Year: 2012
Published on: 01 April, 2012
Page: [485 - 491]
Pages: 7
DOI: 10.2174/092986612800190991
Price: $65

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