Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets

Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10650-4. doi: 10.1073/pnas.91.22.10650.

Abstract

The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • B-Lymphocytes / metabolism
  • Binding Sites
  • Cell Line
  • Consensus Sequence
  • Cytochrome b Group / metabolism
  • DNA Primers
  • Enzyme Activation
  • Genes, src
  • Granulomatous Disease, Chronic / genetics
  • Granulomatous Disease, Chronic / metabolism
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NADH, NADPH Oxidoreductases / biosynthesis*
  • NADPH Dehydrogenase / metabolism*
  • NADPH Oxidases
  • Phagocytes / enzymology*
  • Phosphoproteins / metabolism*
  • Point Mutation
  • Polymerase Chain Reaction
  • Proline
  • Protein Binding
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Spodoptera
  • Transfection

Substances

  • Cytochrome b Group
  • DNA Primers
  • Phosphoproteins
  • Recombinant Proteins
  • neutrophil cytosol factor 67K
  • cytochrome b558
  • Proline
  • NADH, NADPH Oxidoreductases
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • NADPH Dehydrogenase