The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation

Itai Pessach, Zeev Shmelzer, Thomas L. Leto, Mary C. Dinauer, Rachel Levy

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4 Scopus citations

Abstract

We have previously established a model of cytosolic phospholipase A 2 (cPLA2)-deficient PLB-985 cells and demonstrated that cPLA2-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91phox (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91phox C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91phox (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA2 protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47 phox and p67phox to the membrane fractions of both stimulated cell types indicated that the gp91phox C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA2 translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91phox provides a docking site for cPLA2 but is not the site of AA-based regulation of oxidase activity.

Original languageEnglish
Pages (from-to)630-639
Number of pages10
JournalJournal of Leukocyte Biology
Volume80
Issue number3
DOIs
StatePublished - 1 Sep 2006

Keywords

  • Arachidonic acid
  • FAD
  • cPLA

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