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

Reactive sulfur species (RSS) modulate protein functions via Spolysulfidation of reactive Cys residues. Here, we report that Ca/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. incubation of CaMKIV with the exogenous RSS donors NaS ( = 2-4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the NaS -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr 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 Thr was decreased upon treatment with either NaS 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-Thr of endogenous CaMKIV was also inhibited by treatment either with NaS or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via Spolysulfidation of its Cys during the response to ER stress.