Abstract

The NADPH oxidase of neutrophils catalyzes the production of 02 from oxygen and NADPH. The enzyme is dormant in the resting cell, but acquires activity when the cell is stimulated with any of a number of agents. During activation p47PH0X, one of the subunits of the oxidase, is extensively phosphorylated and migrates from the cytosol to the membrane where it assists in the assembly of the active oxidase. We propose to study this phosphorylation and its relationship to the activation of the oxidase in a kinasedependent cell-free oxidase activating system. The p47PH0X will be phosphorylated using a number of kinases and the phosphorylated forms of p47PH0X tested for their ability to support oxidase activation. In those phosphorylated forms that support oxidase activation, the locations of the phosphates will be determined. Possible interactions between the phosphorylation targets will be investigated by site-directed mutagenesis. The role of phosphorylation/dephosphorylation in the deactivation of the oxidase will be examined. The possibility that -SH groups participate in oxidase activation will be investigated. The significance of the interaction between p47PH0X and GTP will be studied. Finally, p47PH0X will be crystallized and its structure determined by x-ray analysis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI028479-12
Application #
6373169
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Voulgaropoulou, Frosso
Project Start
1989-07-01
Project End
2005-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
12
Fiscal Year
2001
Total Cost
$398,925
Indirect Cost

  Institution

Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

DeLano, Frank A; Parks, Dale A; Ruedi, Julie M et al. (2006) Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat. Microcirculation 13:551-66
Lopes, Lucia Rossetti; Dagher, Marie-Claire; Gutierrez, Abel et al. (2004) Phosphorylated p40PHOX as a negative regulator of NADPH oxidase. Biochemistry 43:3723-30
Babior, Bernard M (2004) NADPH oxidase. Curr Opin Immunol 16:42-7
Hoyal, Carolyn R; Gutierrez, Abel; Young, Brandon M et al. (2003) Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase. Proc Natl Acad Sci U S A 100:5130-5
Catz, S D; Johnson, J L (2003) BCL-2 in prostate cancer: a minireview. Apoptosis 8:29-37
Dang, Pham My-Chan; Cross, Andrew R; Quinn, Mark T et al. (2002) Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558 II. Proc Natl Acad Sci U S A 99:4262-5
Catz, Sergio D; Johnson, Jennifer L; Babior, Bernard M (2002) The C2A domain of JFC1 binds to 3'-phosphorylated phosphoinositides and directs plasma membrane association in living cells. Proc Natl Acad Sci U S A 99:11652-7
Catz, Sergio D; Babior, Bernard M; Johnson, Jennifer L (2002) JFC1 is transcriptionally activated by nuclear factor-kappaB and up-regulated by tumour necrosis factor alpha in prostate carcinoma cells. Biochem J 367:791-9
Dang, P M; Cross, A R; Babior, B M (2001) Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558. Proc Natl Acad Sci U S A 98:3001-5
Babior, B M (2000) Phagocytes and oxidative stress. Am J Med 109:33-44

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