Reversible Phosphorylation as a Controlling Factor for Sustaining Calcium Oscillations in HeLa Cells: Involvement of Calmodulin-Dependent Kinase II and a Calyculin A-Inhibitable Phosphatase

Abstract

The role of reversible phosphorylation in histamine-induced Ca²⁺ oscillations in HeLa cells has been investigated by using various activators and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, which proved to be a useful and convenient tool. Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kinase II (CaMK II), only CaMK II was essential. When added during oscillations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca²⁺ spike before halting the process. Introduction of the Ca²⁺/calmodulin-independent catalytic domain of CaMK II into the cells forestalled their response to histamine. These results show that intracellular Ca²⁺ cannot oscillate when CaMK II is locked in either the inactive or the stimulated state. External Ca²⁺ electroporated into cells preloaded with the catalytic domains was quickly removed (but not when the cells were pretreated with the endoplasmic reticulum Ca²⁺-ATPase inhibitor, tapsigargin), indicating that the ATP-driven Ca²⁺ pump was somehow activated by CaMK II. Protein phosphatase inhibitors calyculin A and okadaic acid abolished ongoing oscillations and, when added at low concentrations, prolonged the interspike interval. Immunoprecipitation experiments with ³²P_i-labeled cells provided the first evidence that inositol 1,4,5-trisphosphate receptor (IP₃R) was phosphorylated by CaMK II in vivo. The extent of phosphorylation was increased in the presence of histamine, significantly enhanced by calyculin A, and greatly reduced by W-7. Our observations are consistent with the concept that repetitive phosphorylation-dephosphorylation cycles regulating IP₃R and Ca²⁺ pumps are a controlling factor for sustained Ca²⁺ oscillations in HeLa, and possibly other, cells.