Redox regulation of Ca2+/calmodulin-dependent protein kinase IV via oxidation of its active-site cysteine residue

Abstract

We have recently reported that Ca²⁺/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) is inactivated by reactive sulfur species via polysulfidation of the active-site Cys residue. Here, we show that hydrogen peroxide (H₂O₂) limit CaMKIV activity at the same active-site Cys residue through oxidation and downstream signaling in cells. CaMKIV is phosphorylated at Thr¹⁹⁶ by its upstream CaMK kinase (CaMKK), which induces its full activity. In vitro incubation of CaMKIV with H₂O₂ resulted in reversible inhibition of CaMKK-induced phospho-Thr¹⁹⁶ and the consequent inactivation of CaMKIV. In contrast, mutated CaMKIV (C198V) was refractory to the H₂O₂-induced enzyme inhibition. In transfected cells expressing CaMKIV, Ca²⁺ ionophore-induced CaMKIV phosphorylation at Thr¹⁹⁶ was decreased upon treatment with H₂O₂, whereas cells expressing mutant CaMKIV (C198V) were resistant to H₂O₂ treatment. Modification of free thiol with N-ethylmaleimide revealed that Cys¹⁹⁸ in CaMKIV is a target for S-oxidation. Additionally, the Ca²⁺ influx-induced phospho-Thr¹⁹⁶ of endogenous CaMKIV was also inhibited upon treatment with H₂O₂ in Jurkat T-lymphocytes and cerebellar granule cells. Phosphorylation of cyclic AMP response element-binding protein (CREB) at Ser¹³³, which is downstream of CaMKIV, was also decreased upon treatment with H₂O₂. Thus, our results indicate that oxidation stress regulates cellular function by decreasing the activity of CaMKIV through Cys¹⁹⁸ oxidation.

Keywords: Ca(2+)/calmodulin-dependent protein kinase (CaMK); Phosphorylation; Redox regulation; S-oxidation.