In response to certain cytokines and inflammatory mediators, the activity of the neutrophil NADPH oxidase enzyme is primed for enhanced superoxide production when the cells receive a subsequent oxidase-activating stimulus. The relative role of p38 MAPK in the priming and activation processes is incompletely understood. We have developed a 2-step assay that allows the relative contributions of p38 MAPK activity in priming to be distinguished from those involved in oxidase activation. Using this assay, together with in vitro kinase assays and immunochemical studies, we report that p38 MAPK plays a critical role in TNF␣ priming of the human and porcine NADPH oxidase for superoxide production in response to complement-opsonized zymosan (OpZ), but little, if any, role in neutrophil priming by platelet-activating factor (PAF) for OpZ-dependent responses. The OpZ-mediated activation process per se is independent of p38 MAPK activity, in contrast to oxidase activation by fMLP, where 70% of the response is eliminated by p38 MAPK inhibitors regardless of the priming agent. We further report that incubation of neutrophils with TNF␣ results in the p38 MAPK-dependent phosphorylation of a subpopulation of p47 phox and p67 phox molecules, whereas PAF priming results in phosphorylation only of p67 phox . Despite these phosphorylations, TNF␣ priming does not result in significant association of either of these oxidase subunits with neutrophil membranes, demonstrating that the molecular basis for priming does not appear to involve preassembly of the NADPH oxidase holoenzyme/cytochrome complex prior to oxidase activation.The neutrophil NADPH oxidase is a multiprotein enzyme that catalyzes the production of O 2 Ϫ from oxygen using NADPH as an electron donor (1, 2). The O 2 Ϫ is subsequently converted to powerful oxidizing agents such as hypohalides, singlet oxygen, peroxides, and chloramines, which together with proteases and ions, are primarily responsible for neutrophil-mediated killing of microorganisms (2-4). These oxidase products, however, are also highly destructive to nearby tissues (see Refs. 5-7). Consequently, tight temporal and spatial regulation of the oxidase is required so that it is activated only under appropriate circumstances.The NADPH oxidase holoenzyme consists of 6 subunits. In resting cells in which the oxidase is dormant, 4 of the subunits, p47 phox , p67 phox , p40 phox , and the small GTPase Rac2 are localized exclusively in the cytoplasm whereas the remaining two subunits, p22 phox and gp91 phox form a heterodimeric membrane-bound flavocytochrome known as cytochrome b 558 . Exposure of neutrophils to a variety of diverse stimuli results in translocation and docking of the cytosolic subunits with the membrane subunits (3, 8 -19), allowing electrons to flow from p67 phox -bound NADPH to molecular oxygen via an FAD moiety and two heme groups in the membrane-bound cytochrome (20). The subunit translocation and oxidase activation process is complex and requires multisite phosphorylation of the cytosolic subuni...

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b 558 and cytosolic p67 phox , p47 phox , and p40 phox subunits that undergo membrane translocation upon cellular activation. The function of p40 phox , which binds p67 phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40 phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40 phox in Fc␥R-induced oxidase activation, we used immunofluorescence and real-time imaging of Fc␥R-induced phagocytosis. YFP-tagged p67 phox and p40 phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67 phox and p47 phox accumulation on nascent and internalized phagosomes did not require p40 phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40 phox PI3P-binding domain or wortmannin. Translocation of p40 phox to nascent phagosomes required binding to p67 phox but not PI3P, although the loss of PI3P binding reduced p40 phox retention after phagosome internalization. We conclude that p40 phox functions primarily to regulate Fc␥R-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. (Blood. 2008;112:3867-3877) IntroductionPhagocytic leukocytes are the front-line cellular defense against microbial attack, and are mobilized rapidly to the sites of infection where they ingest and kill opsonized microorganisms. The NADPH oxidase complex plays a central role in this process, as its assembly and activation on phagosomal membranes generate superoxide, the precursor of potent microbicidal oxidants. The importance of this enzyme is demonstrated by genetic defects in the NADPH oxidase complex that cause chronic granulomatous disease (CGD), characterized by recurrent severe and potentially lethal bacterial and fungal infections. 1 The NADPH oxidase includes the membrane-integrated flavocytochrome b, composed of gp91 phox and p22 phox , and the cytosolic components p47 phox , p67 phox , p40 phox , and Rac, a Rho-family GTPase, which translocate to flavocytochrome b upon cellular stimulation to activate superoxide production. [2][3][4] Segregation of regulatory components to the cytosol in resting cells facilitates the temporal and spatial regulation of NADPH oxidase activity. The p67 phox subunit is a Rac-GTP effector 2-4 containing a domain that activates electron transport through the flavocytochrome. 5 In resting cells, p67 phox is associated with p40 phox via complementary PB1 (phagocyte oxidase and Bem1p) motifs present in each protein. 2,[6][7][8] p67 phox is also linked to p47 phox via a high-affinity interaction involving an SH3 domain and a proline-rich region, respectively, in the C-termini of...

Summary Small heat-shock proteins (sHSPs) are a widely conserved family of molecular chaperones, all containing a conserved α-crystallin domain flanked by variable N- and C-terminal tails. We report that IbpA and IbpB, the sHSPs of Escherichia coli, are substrates for the AAA+ Lon protease. This ATP-fueled enzyme degraded purified IbpA substantially more slowly than purified IbpB, and we demonstrate that this disparity is a consequence of differences in maximal Lon degradation rates and not in substrate affinity. Interestingly, however, IbpB stimulated Lon degradation of IbpA both in vitro and in vivo. Furthermore, although the variable N- and C-terminal tails of the Ibps were dispensable for proteolytic recognition, these tails contain critical determinants that control the maximal rate of Lon degradation. Finally, we show that E. coli Lon degrades variants of human α-crystallin, indicating that Lon recognizes conserved determinants in the folded α-crystallin domain itself. These results suggest a novel mode for Lon substrate recognition and provide a highly suggestive link between the degradation and sHSP branches of the protein quality-control network.

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40 phox , p47 phox , and p67 phox , and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40 phox remains less well defined than those of p47 phox and p67 phox . We investigated the biological role of p40 phox in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40 phox using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67 phox levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40 phox reduces both the maximal rate and total amount of ROS produced without altering the K M value of the oxidase for NADPH. Using a series of mutants, p47PX-p40 phox chimeras, and deletion constructs, we found that the p40 phox PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67 phox requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40 phox , however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40 phox can increase oxidase activity ϳ2.5-fold above that of wild-type p40 phox but maintain the requirement for PX and SH3 domain function. We present a model where p40 phox translocates p67 phox to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.