Activation of p47PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase: Phosphorylation of Ser-359 or Ser-370 Precedes Phosphorylation at Other Sites and is Required for Activity

• Published in: The Journal of biological chemistry Vol. 273; no. 52; pp. 35147 - 35152
• Main Authors: Johnson, J L, Park, J, Benna, JE, Faust, L P, Inanami, O, Babior, B M
• Format: Journal Article
• Language: English
• Published: 25.12.1998
• ISSN: 0021-9258

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O- sub(2)) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47PHOX becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47PHOX-deficient B lymphoblasts expressing the p47PHOX S359A/S370A or p47 super(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47PHOX as compared with the same cells expressing wild type p47PHOX. In addition, these mutant p47PHOX proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47PHOX to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O- sub(2) production. These findings, plus those from an earlier study, suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47PHOX: Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47PHOX to the membrane at some point after the first phosphorylation takes place.