Selective roles for alpha-PKC in positive signaling for O-(2) 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 O(-)(2) generation. Ca/DG-dependent alpha-PKC and beta-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 alpha-PKC in positive signaling for fMet-Leu-Phe- and PMA-activated O(-)(2) generation was probed using an siRNA strategy in HL60 cells differentiated to a neutrophilic phenotype (dHL60 cells). A selective decrease in alpha-PKC in dHL60 cells attenuated O(-)(2) generation but not degranulation, and reduced ligand-induced phosphorylation of p47phox as previously shown for beta-PKC. However alpha-PKC, unlike beta-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 O(-)(2) generation confirmed that Ca2+ uptake via SOCC was required for O(-)(2) generation. These results indicate that alpha-PKC and beta-PKC are required for optimal O(-)(2) generation, but play different roles in Ca2+ signaling for phagocytic responses such as O(-)(2) generation.
Korchak
Helen M
HM
Department of Pediatrics, University of Pennsylvania School of Medicine, The Joseph Stokes Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. korchak@email.chop.edu
Dorsey
Lindsay B
LB
Li
Haiying
H
Mackie
DeMauri
D
Kilpatrick
Laurie E
LE
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*
D007527:Isoenzymes; D008024:Ligands; D010703:Phorbol Esters; D034741:RNA, Small Interfering; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051571:Protein Kinase C-alpha; D010196:Pancreatic Elastase; D002118:Calcium