title: NOX1 NADPH oxidase regulation by the NOXA1 SH3 domain.
abstract: We investigated the role of the single SH3 domain of NOXA1 in NOX1 NADPH oxidase function using wild-type and mutated NOXA1 and the products of two variant NOXA1 transcripts isolated from CaCo2 cells by reverse transcription polymerase chain reaction. The first variant, NOXA1(trunc), contained a number of point mutations, including A51T, T261A, and a nonsense mutation at position 274. On transfection into K562 cells stably expressing NOX1 and NOXO1, both NOXA1(trunc) and an equivalent truncated wild-type NOXA1(1-273) were expressed as approximately 29-kDa truncated NOXA1 proteins lacking both PB1 and SH3 domains, yet both were as active as wild-type NOXA1 in phorbol-stimulated superoxide generation. Kinetic analysis demonstrated that truncated NOXA1 activated the NOX1 system at an accelerated rate compared with NOXA1. Deletion studies showed that the slower kinetics of wild-type NOXA1 depended primarily on its SH3 domain, suggesting SH3-dependent delay in forming the active NOX1/NOXO1/NOXA1 complex. The second variant, NOXA1(inhib), encoded a protein lacking the activation domain due to absence of exons 5 and 6 but including a heptapeptide (EPDVPLA) SH3 domain insertion resulting from alternative splicing in exon 14. NOXA1(inhib) failed to support superoxide-generating activity and exhibited transdominant inhibition of NOXA1. Insertion of the heptapeptide into the corresponding site in wild-type NOXA1 inhibited its activity by approximately 90%, rendered it a transdominant inhibitor of wild-type NOXA1, and abrogated binding of its SH3 domain to NOXO1 and p47(phox). These studies demonstrate that, in reconstituted NOX1/NOXO1/NOXA1 systems, the NOXA1 SH3 domain is not required for function but, when present, can critically modulate the activity of the enzyme system.
authors.affiliation: Department of Medicine, University of Texas Health Science Center and South Texas Veterans Health Care System, Audie L. Murphy Division, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. valente@uthscsa.edu
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Valente
authors.forename: Amina
authors.initials: A
authors.lastname: El Jamali
authors.forename: Terry Kay
authors.initials: TK
authors.lastname: Epperson
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Gamez
authors.forename: Doran W
authors.initials: DW
authors.lastname: Pearson
authors.forename: Robert A
authors.initials: RA
authors.lastname: Clark
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476420:NOXA1 protein, human; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C060505:neutrophil cytosolic factor 1