Phagocyte NADPH oxidase p67-phox possesses a novel carboxylterminal binding site for the GTPases Rac2 and Cdc42.
Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox.
Faris
S L
SL
Baltimore Veterans Affairs Medical Center and the Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, 21201, USA.
Rinckel
L A
LA
Huang
J
J
Hong
Y R
YR
Kleinberg
M E
ME
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins
D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins