Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox

Biochemistry. 1997 Jun 17;36(24):7474-80. doi: 10.1021/bi9700936.

Abstract

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47phox, an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47phox leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47phox. A similar conformational change occurs when p47phox is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cyanogen Bromide
  • Cysteine / chemistry
  • Enzyme Activation / drug effects
  • Ethylmaleimide / chemistry
  • Humans
  • Molecular Sequence Data
  • NADPH Oxidases
  • Neutrophils / enzymology*
  • Peptide Fragments / chemistry
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Conformation*
  • Protein Kinase C / pharmacology*
  • Structure-Activity Relationship

Substances

  • Peptide Fragments
  • Phosphoproteins
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Protein Kinase C
  • Cysteine
  • Ethylmaleimide
  • Cyanogen Bromide