p40phox Down-regulates NADPH Oxidase Activity through Interactions with Its SH3 Domain

doi:10.1074/jbc.272.14.9141

p40^phox Down-regulates NADPH Oxidase Activity through Interactions with Its SH3 Domain*

From the Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20893

^‡ To whom correspondence should be addressed:
Building 10, Rm. 11 N 106, NIAID, National Institutes of Health, Bethesda, MD 20892.
Tel.: 301-402-5120; Fax: 301-402-4369; E-mail: tleto{at}atlas.niaid.nih.gov

Abstract

The NADPH oxidase of phagocytes generates microbicidal oxidants in response to a variety of stimuli. Its activation and assembly involve multiple SH3 domain interactions among several oxidase components. Here we present evidence that the cytosolic oxidase-associated protein, p40^phox, mediates down-regulation of NADPH oxidase through interactions with its SH3 domain. Recombinant p40^phox was produced in several eukaryotic expression systems (insect, mammalian, and yeast) to explore its role in oxidase function in relation to domains involved in interactions with other factors, p47^phox and p67^phox. p40^phox inhibited oxidase activity in vitro when added to neutrophil membranes and recombinant p47^phox, p67^phox, and p21rac. Co-transfection of p40^phox into K562 cells resulted in significant decreases (∼40%) in whole cell oxidase activity. Furthermore, the isolated SH3 domain of p40^phox was even more effective in inhibiting whole cell oxidase activity, consistent with experiments showing that this domain binds to the same proline-rich target in p47^phox (residues 358-390) that interacts with p67^phox. In contrast, deletion of the carboxyl-terminal domain of p40^phox that binds to p67^phox did not relieve its oxidase inhibitory effects. Thus, p40^phox appears to down-regulate oxidase function by competing with an SH3 domain interaction between other essential oxidase components.

Footnotes

↵* 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.
↵¹ The abbreviations used are:

CGD

chronic granulomatous disease

SH3

Src homology 3

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

PMA

phorbol 12-myristate 13-acetate
- Received August 27, 1996.
- Revision received January 16, 1997.