A fluorescently tagged C-terminal fragment of p47phox detects NADPH oxidase dynamics during phagocytosis.
The assembly of cytosolic p47(phox) and p67(phox) with flavocytochrome b(558) 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 b(558)-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.
Li
Xing Jun
XJ
Department of Pediatrics (Hematology/Oncology), Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Tian
Wei
W
Stull
Natalie D
ND
Grinstein
Sergio
S
Atkinson
Simon
S
Dinauer
Mary C
MC
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003573:Cytochrome b Group / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D019255:NADPH Oxidases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D055232:Proline-Rich Protein Domains; D021381:Protein Transport
D003573:Cytochrome b Group; D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; C055525:phosphatidylinositol 3-phosphate; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1