Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

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 approximately 2.5-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.

MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation.

The cellular response to DNA damage is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 protein phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. To investigate DNA damage responses mediated by the p38/stress-activated protein kinase (SAPK) axis of signaling, the optimal phosphorylation motifs of mammalian p38alpha SAPK and MAPKAP kinase-2 were determined. The optimal substrate motif for MAPKAP kinase-2, but not for p38 SAPK, closely matches the 14-3-3 binding site on Cdc25B/C. We show that MAPKAP kinase-2 is directly responsible for Cdc25B/C phosphorylation and 14-3-3 binding in vitro and in response to UV-induced DNA damage within mammalian cells. Downregulation of MAPKAP kinase-2 eliminates DNA damage-induced G2/M, G1, and intra S phase checkpoints. We propose that MAPKAP kinase-2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells.

Distinct ligand-dependent roles for p38 MAPK in priming and activation of the neutrophil NADPH oxidase.

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 TNFalpha 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 TNFalpha 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, TNFalpha 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.

A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC delta in intracellular production of reactive oxygen species by the NADPH oxidase.

Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type. Using this system, we biochemically dissect molecular events and signaling pathways involved in NADPH oxidase assembly and demonstrate specific roles for PKC delta, PI(3,4)P(2)/PI(3,4,5)P(3), and PI(3)P in the PMA-dependent intracellular activation process. This system should be of great utility for the study of cell signaling events that regulate the intracellular production of reactive oxygen species by neutrophils.

Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii.

A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii. The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of approximately 80-90 kDa. Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a approximately 20 kDa species, suggesting it is an oligomer. The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC. Additionally, monomers of alcohol oxidase (AO), an abundant protein of C. boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease. Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.