THE PHOSPHORYLATION OF THE RESPIRATORY BURST OXIDASE COMPONENT P47(PHOX) DURING NEUTROPHIL ACTIVATION - PHOSPHORYLATION OF SITES RECOGNIZEDBY PROTEIN-KINASE-C AND BY PROLINE-DIRECTED KINASES

The respiratory burst oxidase catalyzes the production of O-2(radical anion) from oxygen and NADPH. It is dormant in resting cells but becom es active when the cells are stimulated, Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase compo nent p47(phox), which moves from cytosol to the membrane during oxidas e activation. Using immunopurified p47(phox) isolated from P-32(i)-loa ded neutrophils activated with phorbol myristate acetate, we showed th at all the P-32 was in the C-terminal CNBr fragment of the protein, an d that in that fragment, Ser-303, Ser-304, Ser-320, Ser328, Ser-345, a nd Ser-348 and at least one of the three serines, Ser-359, Ser-370, an d Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and S er-388 were not, Of the phosphorylated serines, Ser-303, Ser-304, Ser3 20, and Ser-328 are located in protein kinase C substrate sequences. S er-345 and Ser-348, however, are located in sequences recognized by mi togen-activated protein (MAP) kinase (-PXSP-). This finding suggests t hat MAP kinase or a related proline-directed kinase may participate in the regulation of O-2(radical anion) production by activated neutroph ils. The tryptic peptide map of p47(phox) phosphopeptides from neutrop hils activated by N-formyl-methionyl-leucyl-phenylalanine closely rese mbled that of p47(phox) phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to ea ch agent.