This is a revision of a competing renewal proposal to study mechanisms of activation of the respiratory burst oxidase of phagocytes. The Applicant was one of the developers of the first cell- free assay system for activation of NADPH oxidase activity, an assay system that led to much of the molecular identification of components of the oxidase to date. In more recent years, recognizing that the original cell-free activation scheme did not require ATP or protein phosphorylation, the Applicant developed a novel cell-free activation scheme involving phosphatidic acid, diacylglycerol and ATP in conjunction with neutrophil cytosol and membranes. Based on the Applicant's studies of phospholipase D activation and phosphatidic acid production (PA) during the activation of the NADPH oxidase, the applicant developed the current cell-free activation scheme of the oxidase by PA plus diacylglycerol (DG), indicating that the products of phospholipase D activity are able to activate the oxidase. In contrast to the SDS/DG system, in which protein kinase inhibitors had no effect on either cell-free activation or activity, NADPH oxidase activity activated in the cell-free PA/DG system was inhibited by 75% by inclusion of protein kinase inhibitors such as staurosporine and H-7. ATP enhances the activity of the system, whereas non-hydrolyzable ATP analogs do not, but the inclusion of GTP-g-S also enhances the activity of the system, but not as much as ATP plus GTP-g-S. The Applicant has been able to show that the system results in phosphorylation of p47phox and that p47 phosphorylation precedes oxidase activity. The identity of p47 as one of the phosphorylated proteins in this system was confirmed by performing the experiment with cytosol from a patient with p47 phox deficiency, in which no p47 phosphorylation was detected, and in the same sample when exogenous recombinant p47 was present, in which case it was phosphorylated. Taken together, these and other data form the basis of the new specific aims. The first is to purify, clone and characterize the PA-activated protein kinase that phosphorylates recombinant p47phox, and the second to determine the physiological relevance of the PA-activated protein kinase for NADPH oxidase activation. The Applicant has developed a screening assay for purification of the PA-activated protein kinase in which recombinant p47 is phosphorylated in the presence of 32P-g-ATP. The source of the protein substrate is the supernatant of baculovirus infected Sf9 cells or expressed in bacteria as a GST fusion protein, both constructs being provided by Tom Leto, with whom the applicant collaborates. Following purification by standard techniques with the collaboration of Dr. Reidar Wallin who has extensive protein purification expertise, sequence will be obtained on either the whole protein or tryptic or cyanogen bromide fragments, and eventually cloned using antibodies derived from sequence to screen protein-expressing libraries. The phosphopeptide mapping will be performed first on PA-activated protein kinase in the cell free system using recombinant p47phox, and then the patterns verified in the intact cell. The phosphorylation site(s) will then direct the next part of the proposal, to create and test site-directed mutagenized forms of p47phox to determine the requirement of individual phosphorylation sites in p47phox for activation of NADPH oxidase the cell-free system and in intact cells. The mutagenized p47phox constructs will be expressed in Sf9 insect cells and purified for use in the cell-free system. Selected mutant CDNAS will be incorporated into mammalian vectors for transfection into p47phox- deficient cell lines, specifically in a K562 cell line model missing only p47 and p67, developed by Dr. Tom Leto, a cell line model that generates 10-fold more superoxide than the EBV-transformed lymphocyte systems. An additional aim is to fully reconstitute the superoxide generating system using purified PA-activated protein kinase with purified/recombinant NADPH oxidase components. The purified cytochrome b558 will be donated by Dr. Jesaitis and Quinn for these studies. Lastly, the Applicant will determine whether phosphorylation of p47phox by the PA-activated protein kinase induces assembly of NADPH oxidase via the SH3 domains of p47phox and p67phox. This will be done in collaboration with Dr. Leto, in an assay in which GST-p22phox fusion proteins are applied to nitrocellulose, and the amount of p47phox bound before and after phosphorylation with PA-activated protein kinase determined using an anti-p47phox antibody.
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