NOX1 NADPH oxidase regulation by the NOXA1 SH3 domain

Anthony J. Valente, Amina El Jamali, Terry Kay Epperson, Maria J. Gamez, Doran W. Pearson, Robert A. Clark

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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, NOXA1trunc, 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 NOXA1trunc and an equivalent truncated wild-type NOXA1(1-273) were expressed as ∼ 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, NOXA1inhib, 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. NOXA1inhib 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 ∼ 90%, rendered it a transdominant inhibitor of wild-type NOXA1, and abrogated binding of its SH3 domain to NOXO1 and p47phox. 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.

Original languageEnglish (US)
Pages (from-to)384-396
Number of pages13
JournalFree Radical Biology and Medicine
Volume43
Issue number3
DOIs
StatePublished - Aug 1 2007

Keywords

  • NADPH oxidase
  • NOX1
  • NOXA1
  • SH3 domain
  • Superoxide

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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