The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases.
The respiratory burst oxidase catalyzes the production of O2.- from oxygen and NADPH. It is dormant in resting cells but becomes active when the cells are stimulated. Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase component p47phox, which moves from cytosol to the membrane during oxidase activation. Using immunopurified p47phox isolated from 32Pi-loaded neutrophils activated with phorbol myristate acetate, we showed that all the 32P was in the C-terminal CNBr fragment of the protein, and that in that fragment, Ser-303, Ser-304, Ser-320, Ser-328, Ser-345, and Ser-348 and at least one of the three serines, Ser-359, Ser-370, and Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and Ser-388 were not. Of the phosphorylated serines, Ser-303, Ser-304, Ser-320, and Ser-328 are located in protein kinase C substrate sequences. Ser-345 and Ser-348, however, are located in sequences recognized by mitogen-activated protein (MAP) kinase (-PXSP-). This finding suggests that MAP kinase or a related proline-directed kinase may participate in the regulation of O2.- production by activated neutrophils. The tryptic peptide map of p47phox phosphopeptides from neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine closely resembled that of p47phox phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to each agent.
el Benna
J
J
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Faust
L P
LP
Babior
B M
BM
D000595:Amino Acid Sequence; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D038461:Proline-Directed Protein Kinases; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010768:Phosphoserine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate