| First Author | Valente AJ | Year | 2007 |
| Journal | Free Radic Biol Med | Volume | 43 |
| Issue | 3 | Pages | 384-96 |
| PubMed ID | 17602954 | Mgi Jnum | J:122814 |
| Mgi Id | MGI:3715561 | Doi | 10.1016/j.freeradbiomed.2007.04.022 |
| Citation | Valente AJ, et al. (2007) NOX1 NADPH oxidase regulation by the NOXA1 SH3 domain. Free Radic Biol Med 43(3):384-96 |
| abstractText | 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. |
