Selective roles for α-PKC in positive signaling for O2− generation and calcium mobilization but not elastase release in differentiated HL60 cells

Protein kinase C (PKC) isotypes and Ca2+ mobilization have been implicated in phagocytic cell functions such as O2− generation. Ca/DG-dependent α-PKC and β-PKC have similar substrate specificities and cofactor requirements in vitro. However it is not known if these isotypes play redundant or unique roles in the intact cell. In the present study, a role for α-PKC in positive signaling for fMet-Leu-Phe- and PMA-activated O2− generation was probed using an siRNA strategy in HL60 cells differentiated to a neutrophilic phenotype (dHL60 cells). A selective decrease in α-PKC in dHL60 cells attenuated O2− generation but not degranulation, and reduced ligand-induced phosphorylation of p47phox as previously shown for β-PKC. However α-PKC, unlike β-PKC, was a positive regulator of fMet-Leu-Phe-triggered Ca2+ uptake via SOCC (Store Operated Calcium Channels). The ability of a selective SOCC inhibitor, MRS1845, to decrease fMet-Leu-Phe induced Ca2+ uptake and O2− generation confirmed that Ca2+ uptake via SOCC was required for O2− generation. These results indicate that α-PKC and β-PKC are required for optimal O2− generation, but play different roles in Ca2+ signaling for phagocytic responses such as O2− generation.