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, 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 gamma-phosphoryl transfer, are inactive. 4) The ability of ATP to support GEF-induced oxidase activation is explained by ATP serving as a gamma-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.
Mizrahi
Ariel
A
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, PO Box 39040, Tel Aviv 69978, Israel.
Molshanski-Mor
Shahar
S
Weinbaum
Carolyn
C
Zheng
Yi
Y
Hirshberg
Miriam
M
Pick
Edgar
E
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D019255:NADPH Oxidases / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017368:Protein Prenylation; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; D009701:Nucleoside-Diphosphate Kinase; D020830:rac1 GTP-Binding Protein