Polymorphonuclear neutrophils (PMN) mediate not only host surveillance and defense but also inflammatory and other injurious reactions. Chemotactic factors (CF) guide PMN in these capacities. They bind to PMN receptors that issue intracellular signals that regulate key response-eliciting elements. We will investigate one family of such elements, the protein kinases C (PKC). We will also examine the signals derived from phospholipids (PL) that direct PKC to move from a latent state in cytosol to an active membrane-associated kinase. This translocation is regarded as secondary to a Ca2+ transient signal. In PMN, however, CF stimulate PKC movements comprised of both Ca2+ transient-dependent and Ca2+ transient-independent components. In contrast, 5-oxo-eicosatetraenoate causes only Ca2+ transient-dependent and nanomolar levels of arachidonic acid (AA) induce mainly Ca2+ transient-independent PKC movements. We hypothesize that the various PKC isoforms in PMN manifest different patterns of translocation in response to CF, 5-oxo- eicosatetraenoate, and AA; that PMN stimulated with CF issue Ca2+-transient-independent translocation signals; and that AA is one such signal. We will track PKC isoforms in PMN; determine the mechanism of AA's effect on PKC; examine the Ca2+-transient requirements for PMN to metabolize PL into signals for PKC translocation; and define the role AA and other lipid-derived signals play in mediating PKC translocation as well as cell function. Systems for experimentation include human PMN; HL-60 cells reversibly depleted of AA; HEK 293 cells stably expressing wild type or mutant PKC fused to a fluorescent protein; the latter cells transiently expressing a CF receptor; and a cell- free model. We will track PKC by Western blots and fluorescent laser confocal microscopy; measure the metabolism of PL to AA, phosphatidyl-inositols, diacylglycerol, and phosphatidic acid by mass; develop constructs encoding single site, deletion, and truncated PKC species by mutagenesis; and use these constructs to define the structural domains and domain functions in PKC required for PKC to respond to AA or CF. We will test the relevancy of these biochemical studies on PMN and HL-60 cells in assays of superoxide production and other responses. Results of this work will apply to all mammalian cells, to stimuli that, like CF, act on serpentine receptors, and to the many regulatory proteins with structural homology to PKC. The studies should reveal a framework in which to view PKC regulation, one where Ca2+ transients and Ca2+-independent signals (e.g. AA) issue from cells to direct the movement of different PKC isoforms. By making this link between PKC and AA, our studies will suggest new pharmacological and nutritional strategies for the abatement of PKC translocation and thereby the untoward cellular responses which cause self-injury and other pathological reactions.
| Wooten, Rhonda E; Willingham, Mark C; Daniel, Larry W et al. (2008) Novel translocation responses of cytosolic phospholipase A2alpha fluorescent proteins. Biochim Biophys Acta 1783:1544-50 |
