NOX1 NADPH oxidase regulation by the NOXA1 SH3 domain

We investigated the role of the single SH3 domain of NOXA I in NOX I NADPH oxidase function using wild-type and mutated NOXA I 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 NOXAI(1-273) were expressed as similar to 29-kDa truncated NOXA I proteins lacking both PB I and SH3 domains, yet both were as active as wild-type NOXA I in phorbol-stimulated superoxide generation. Kinetic analysis demonstrated that truncated NOXAI activated the NOX1 system at an accelerated rate compared with NOXAL Deletion studies showed that the slower kinetics of wild-type NOXA I 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 NOXAI inhibited its activity by similar to 90%, rendered it a transdominant inhibitor of wild-type NOXAI, and abrogated binding of its SH3 domain to NOXO1 and p47(phox). These studies demonstrate that, in reconstituted NOX1/NOXO1/NOXA1 systems, the NOXA I SH3 domain is not required for function but, when present, can critically modulate the activity of the enzyme system. (C) 2007 Elsevier Inc. All rights reserved.