Reactive sulfur species inactivate Ca²⁺/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue

Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca²⁺/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr¹⁹⁶ by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na₂S_n (n = 2–4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr¹⁹⁶ and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na₂S_n-induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys¹⁹⁸ in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr¹⁹⁶ and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr¹⁹⁶ was decreased upon treatment with either Na₂S₄ or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr¹⁹⁶ of endogenous CaMKIV was also inhibited by treatment either with Na₂S₄ or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S-polysulfidation of its Cys¹⁹⁸ during the response to ER stress.