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Vol. 20, Issue 5, 1520-1532, March 1, 2009

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A Fluorescently Tagged C-Terminal Fragment of p47^phox Detects NADPH Oxidase Dynamics during Phagocytosis

Xing Jun Li^*, Wei Tian^*, Natalie D. Stull^*, Sergio Grinstein, Simon Atkinson, and Mary C. Dinauer^*,,||

*Department of Pediatrics (Hematology/Oncology), Herman B Wells Center for Pediatric Research, Riley Hospital for Children, and Departments of Microbiology/Immunology, ^||Medical and Molecular Genetics, and Medicine (Nephrology), Indiana University School of Medicine, Indianapolis, IN 46202; and Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Submitted June 19, 2008; Revised November 7, 2008; Accepted December 24, 2008
Monitoring Editor: Jennifer Lippincott-Schwartz

The assembly of cytosolic p47^phox and p67^phox with flavocytochrome b₅₅₈ at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47^phox and p67^phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67^phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67^phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47^phox PRR. This fragment was recruited in a flavocytochrome b₅₅₈-dependent, p67^phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67^phox SH3b domain and suggest that the p47^phox/p67^phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67^phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.

Author contributions: X.J.L. designed, performed and analyzed experiments, prepared the figures, and drafted the manuscript. W.T. helped to set up the live imaging system for analysis of NADPH oxidase assembly during phagocytosis, N.S. helped with retrovirus transductions in PLB-985 cells, and S.G. and S.A. helped with experimental design and interpretation of the data. M.C.D. oversaw this entire project including the experimental design, analysis, and interpretation of the data, and preparation of the manuscript.

Address correspondence to: Mary C. Dinauer (mdinauer{at}iupui.edu)

Abbreviations used: aa, amino acid; CGD, chronic granulomatous disease; DCF, dichlorodihydrofluorescein; IgG-Zym, IgG-opsonized zymosan; O₂^–, superoxide; PB1, Phox and Bem1; PI(3)P, phosphatidylinositol 3-phosphate; PRR, proline-rich region; PX, PhoX domain; SH3, Src homology 3; SOD, superoxide dismutase.

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