Mapping the Domains of Interaction of p40 with Both p47 and p67 of the Neutrophil Oxidase Complex Using the Two-hybrid System

doi:10.1074/jbc.270.11.5695

Mapping the Domains of Interaction of p40 with Both p47 and p67 of the Neutrophil Oxidase Complex Using the Two-hybrid System (*)

From the Commissariat à l'Energie Atomique/Laboratoire de Biochimie (URA 1130/CNRS), Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France

^§ To whom correspondence should be addressed. Tel.: 33 76 88 34 76; Fax: 33 76 88 51 85.

Abstract

The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67, p47 and p40 as well as a small G protein Rac (for review, see (1, 2, 3)). A truncated form of the p40 cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47 as target. This truncated form of p40 consisting of the Src Homology 3 (SH3) domain to the 3′ stop codon was also shown to interact with p67 in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47 or p67. Results show that the SH3 domain of p40 is sufficient for interaction with p47, whereas the C terminus of p40 but not its SH3 domain is involved in the interaction with p67.

Footnotes

↵* This work was supported in part by grants from the Centre National de la Recherche Scientifique, from the Association pour la Recherche sur le Cancer, and from the Université Joseph Fourier-Faculté de Médecine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SH3

Src homology domain 3

LB-EBV

B lymphocyte cells immortalized by the Epstein-Barr virus

bp

base pair(s)

PCR

polymerase chain reaction

X-gal

5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
↵² L. Van Aelst, unpublished results.
- Received December 21, 1994.
- Revision received January 20, 1995.