Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes.
Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.
Bey
Erik A
EA
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Xu
Bo
B
Bhattacharjee
Ashish
A
Oldfield
Claudine M
CM
Zhao
Xiaoxian
X
Li
Qing
Q
Subbulakshmi
Venkita
V
Feldman
Gerald M
GM
Wientjes
Frans B
FB
Cathcart
Martha K
MK
D000098:Acetophenones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex / Q000378:metabolism; D016718:Arachidonic Acid / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D004789:Enzyme Activation / Q000276:immunology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008262:Macrophage Activation / Q000276:immunology*; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology* / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism
D000098:Acetophenones; D000936:Antigen-Antibody Complex; D001578:Benzopyrans; D007211:Indoles; D008301:Maleimides; D009281:Naphthalenes; D020319:Oligodeoxyribonucleotides, Antisense; D010750:Phosphoproteins; D020033:Protein Isoforms; D013481:Superoxides; D016718:Arachidonic Acid; C085746:rottlerin; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C058819:calphostin C; C070515:bisindolylmaleimide I