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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021961-11
Application #
2061657
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-12-01
Project End
1997-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Gabig, T G; Crean, C D; Mantel, P L et al. (1995) Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation. Blood 85:804-11
Graves, V; Gabig, T; McCarthy, L et al. (1992) Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils. Blood 80:776-87
Eklund, E A; Marshall, M; Gibbs, J B et al. (1991) Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein. J Biol Chem 266:13964-70
Eklund, E A; Gabig, T G (1990) Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2- -generating NADPH oxidase. J Biol Chem 265:8426-30
Broxmeyer, H E; Cooper, S; Gabig, T (1989) The effects of oxidizing species derived from molecular oxygen on the proliferation in vitro of human granulocyte-macrophage progenitor cells. Ann N Y Acad Sci 554:177-84
Parkinson, J F; Gabig, T G (1988) Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations. J Biol Chem 263:8859-63
Akard, L P; English, D; Gabig, T G (1988) Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst. Blood 72:322-7
English, D; Debono, D J; Gabig, T G (1987) Relationship of phosphatidylinositol bisphosphate hydrolysis to calcium mobilization and functional activation in fluoride-treated neutrophils. J Clin Invest 80:145-53
Gabig, T G; English, D; Akard, L P et al. (1987) Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. J Biol Chem 262:1685-90
English, D; Gabig, T G (1986) Differentiation of cellular processes involved in the induction and maintenance of stimulated neutrophil adherence. Blood 67:1314-22

Showing the most recent 10 out of 11 publications