Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst componentp47(phox)

The phosphorylation of p47(phox) is widely viewed as an important step in t he activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by eithe r PMA or formylmethionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presenc e of previously suggested kinases, including protein kinase C isotypes, mit ogen-activated protein kinase and protein kinase B. Gel filtration and rena turation in substrate gels suggest a molecular mass of between 45 and 51 kD a. The kinase activity was independent of calcium and lipids but was potent ly inhibited by staurosporine. Treatment with protein phosphatase 2Ac sugge sted that the kinase was activated by serine/threonine phosphorylation. Pho sphopeptide maps indicated that the kinase phosphorylated p47(phox) on simi lar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kina se that may play a part in the regulation of neutrophil NADPH oxidase throu gh its ability to phosphorylate p47(phox).