Important microbicidal and cytotoxic functions of phagocytic cells rely on reactive oxidants derived from the membrane-associated NADPH oxidase. The enzymatic control of 02- production by these cells is tightly regulated. Our studies of cellfree NADPH oxidase activation have recently identified a ras-family GTPase protein, Krev-1 (rapla), as an essential activator of the NADPH oxidase. This recombinant rapla-reconstituted cellfree system has now emerged as the only biochemical system suitable to study the direct physical interaction of a ras-family GTPase with an identified effector enzyme (NADPH oxidase cytochrome b558). Therefore a major focus of the proposed studies will be to characterize the structural requirements for rapla function in this system.
The aims are: (1) to determine the essential primary and deduced secondary structural features of rapla GTPase required for function in NADPH oxidase activation (2) to examine the role of protein kinase A-mediated phosphorylation of rapla Serl80 in downregulation of NADPH oxidase and (3) to further define the mechanism of action of the lipid thiobis-deactivator of NADPH oxidase, thereby testing the hypothesis that the deactivator species displaces endogenous geranylgeranylated rapla from the active NADPH oxidase catalytic unit. The rapla structure-function studies will utilize a series of chimeric ras/rapla recombinant proteins as well as recombinant rapla proteins with specific point mutations that have been shown to alter critical properties of guanine nucleotide binding/GTP hydrolysis, or to alter function in vivo in assays of suppression-of-transformation by rapla. Such studies are ideally suited for this cellfree system since we have shown that cellfree NADPH oxidase activation is fully dependent on rapla, whereas ras proteins are totally inactive in this system. The proposed studies should provide detailed information regarding the biochemical function of a ras-family GTPase protein, rapla.
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