Differential Kinetics and Inhibition of Purified Recombinant Tyrosine Kinase 2 (TYK-2) and Its Catalytic Domain JH-1
2012
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.
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