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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1105684

Received for publication November 24, 2005.

Accepted for publication April 4, 2006.

Article

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 {dagger}, Mary C. Dinauer {ddagger}, and Rachel Levy *@

*Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva, Israel; {dagger}Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland; and {ddagger}Herman B. Wells Center for Pediatric Research and Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis

@ To whom correspondence should be addressed. E-mail: ral{at}bgumail.bgu.ac.il.


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Abstract

We have previously established a model of cytosolic phospholipase A2 (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 p47phox 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.

Key Words: FAD • arachidonic acid