Activation of the Phagocyte NADPH Oxidase by Rac Guanine Nucleotide Exchange Factors in Conjunction with ATP and Nucleoside Diphosphate Kinase

Activation of the phagocyte NADPH oxidase is the consequence of the assembly of membranal cytochrome b559 with the cytosolic components p47phox, p67phox, and the GTPase Rac and is mimicked by a cell-free system comprising these components and an activator. We designed a variant of this system, consi...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2005-02, Vol.280 (5), p.3802-3811
Main Authors: Mizrahi, Ariel, Molshanski-Mor, Shahar, Weinbaum, Carolyn, Zheng, Yi, Hirshberg, Miriam, Pick, Edgar
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Animals
Cell Membrane - enzymology
Escherichia coli - enzymology
Guanine Nucleotide Exchange Factors - metabolism
Guanosine Diphosphate - metabolism
Guanosine Triphosphate - metabolism
Guinea Pigs
Macrophages, Peritoneal - enzymology
NADPH Oxidases - metabolism
Nucleoside-Diphosphate Kinase - metabolism
Phagocytosis - physiology
Phosphoproteins - metabolism
Protein Binding - physiology
Protein Prenylation
rac1 GTP-Binding Protein - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Activation of the phagocyte NADPH oxidase is the consequence of the assembly of membranal cytochrome b559 with the cytosolic components p47phox, p67phox, and the GTPase Rac and is mimicked by a cell-free system comprising these components and an activator. We designed a variant of this system, consisting of membranes, p67phox, prenylated Rac1-GDP, and the Rac-specific guanine nucleotide exchange factor (GEF) Trio, in which oxidase activation is induced in the absence of an activator and p47phox. We now show that: 1) Trio and another Rac GEF (Tiam1) act by inducing GDP to GTP exchange on prenylated Rac1-GDP and that our earlier assertion that activation is GTP-independent is explained by contamination of p67phox preparations with GTP and/or ATP. 2) Oxidase activation by Rac GEFs is supported not only by GTP but also by ATP. 3) Non-hydrolysable GTP analogs are active, whereas ATP analogs, incapable of γ-phosphoryl transfer, are inactive. 4) The ability of ATP to support GEF-induced oxidase activation is explained by ATP serving as a γ-phosphoryl donor for a membrane-localized nucleoside diphosphate kinase (NDPK), converting GDP to GTP. 5) The existence of a NDPK in macrophage membranes is proven by functional, enzymatic, and immunologic criteria. 6) NDPK acts on free GDP, and the newly formed GTP is bound again to Rac. 7) Free GDP is derived exclusively by dissociation from prenylated Rac1-GDP, mediated by GEF. NDPK and GEF appear to be functionally linked in the sense that the availability of GDP, serving as substrate for NDPK, is dependent on the level of activity of GEF.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M410257200