The active-site cysteine residue of Ca/calmodulin-dependent protein kinase I is protected from irreversible modification via generation of polysulfidation.

Ca/calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys by RSS. In vitro incubation of CaMKI with the exogenous RSS donor NaS resulted in a dose-dependent inhibition of the phosphorylation at Thr by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the NaS-induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with NaS, whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys by RSS, thereby protecting it from irreversible modification.