The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells.
The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67phox, p47phox and p40phox which translocate to the membrane upon activation. Known mechanisms underlying the translocation of these proteins include polyphosphorylation of p47phox and specific Src homology 3/polyproline motif interactions. In this study, through two-dimensionnal electrophoresis and immunoprecipitation experiments, we show using dimethylsulfoxide-differentiated HL60 promyelocytes that p40phox is in a basal phosphorylated state in resting cells and undergoes further phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol myristate acetate or by the formyl peptide fMet-Leu-Phe-Lys. Moreover, the extent of phosphorylation is strongly correlated with the level of superoxide production. Typically, in cells transiently activated by fMet-Leu-Phe-Lys, onset of superoxide production coincides with the appearance of new phosphorylated species of p40phox and, at the end of the respiratory burst, dephosphorylation of p40phox is observed. In vitro assays show that the kinase(s) involved in the phosphorylation of p40phox differ from those which participate in the phosphorylation of p47phox. This suggests that, in the cell, the phosphorylation of p40phox and of p47phox are under the control of two different kinase pathways.
Fuchs
A
A
Laboratoire BBSI (UMR 314/CNRS), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Bouin
A P
AP
Rabilloud
T
T
Vignais
P V
PV
D002454:Cell Differentiation; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D018922:HL-60 Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D008956:Models, Chemical; D008970:Molecular Weight; D019255:NADPH Oxidases / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013463:Sulfuric Acid Esters; C085072:neutrophil cytosol factor 40K; D019255:NADPH Oxidases; C007482:dimethyl sulfate; D013755:Tetradecanoylphorbol Acetate