Kinase-dependent Activation of the Leukocyte NADPH Oxidase in a Cell-free System


  1. Bernard M. Babior**
  1. From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, the
  2. Department of Biochemistry, Kyungpook National University, Taegu, Korea, and
  3. § INSERM U-294 at CHU X. Bichat, Paris, France
  1. **To whom correspondence should be addressed.


The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2 at the expense of NADPH: 2 O2 + NADPH → 2 O2 + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47PHOX, an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47PHOX. The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47PHOX and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47PHOX phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47PHOX is more physiological than activation by amphiphiles, because the mutant p47PHOX S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.


  • § Visiting Investigator from the Dept. of Biochemistry, Kyungpook National University, Taegu, Korea.

  • Chargé de Recherche-1-CNRS and the recipient of a postdoctoral fellowship from the Arthritis Foundation.

  • * This work was supported in part by United States Public Health Service Grants AI-24227, AI-28479, and RR-00833, the Stein Endowment Fund, and Basic Science Research Institute Grant BSRI-4403 from the Ministry of Education of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Received June 6, 1996.
  • Revision received February 6, 1997.
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