Multiple cysteine residues are implicated in Janus kinase 2-mediated catalysis.
The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Naila M
NM
Mamoon
John K
JK
Smith
Kiranam
K
Chatti
Sheeyong
S
Lee
Kanakadurga
K
Kundrapu
Roy J
RJ
Duhé
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D047428:Protein Kinase Inhibitors; D053614:Janus Kinase 2; D003545:Cysteine