Reversible inactivation of Ca2+/calmodulin-dependent protein kinase IV by oxidation of the active-site cysteine residue

Abstract

Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) is serine/threonine-specific protein kinase that is involved in many signaling cascades, and plays a key role in memory. CaMKIV is activated via binding to Ca 2+ /CaM complex and phosphorylation by upstream kinase, CaM kinase. In the present study, we investigate direct mechanism of inactivation of CaMKIV by oxidative stress. We show that CaMKIV is directly inhibited by its sulfenic acid formation at the Cys198. In situ studies demonstrated that treatment of CaMKIV with hydrogen peroxide (H 2 O 2 ) results in inactivation of the enzyme, with a concomitant sulfenic acid formation of CaMKIV at Cys198. The methodology of detection of sulfenic acid formation is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Mutagenesis studies confirmed that sulfenic acid formation of Cys198 is both necessary and sufficient for the inhibition of CaMKIV by H 2 O 2 . In transfected cells expressing CaMKIV, treatment with H 2 O 2 caused a reversible decrease in CaMKIV activity. Cells expressing mutant CaMKIV (C198V) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKIV via its sulfenic acid formation at Cys198 is an important mechanism in processing calcium signal transduction in cells.