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 Blood, 1 December 2005, Vol. 106, No. 12, pp. 3962-3969.
Prepublished online as a Blood First Edition Paper on August 11, 2005; DOI 10.1182/blood-2005-03-0859.

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PHAGOCYTES

p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils

Kendra D. Martyn, Moon-Ju Kim, Mark T. Quinn, Mary C. Dinauer, and Ulla G. Knaus

From the Department of Immunology, The Scripps Research Institute, La Jolla, CA; the Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology) and Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, IN; and the Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT.

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays an instrumental role in host defense and contributes to microbicial killing by releasing highly reactive oxygen species. This multicomponent enzyme is composed of membrane and cytosolic components that assemble in the plasma membrane or phagolysosome. While the guanosine S'-triphosphatase (GTPase) Rac2 has been shown to be a critical regulator of NADPH oxidase activity and assembly, the role of its effector, p21-activated kinase (Pak), in oxidase function has not been well defined. Using HIV-1 Tat-mediated protein transduction of Pak inhibitory domain, we show here that Pak activity is indeed required for efficient superoxide generation in intact neutrophils. Furthermore, we show that Pak translocates to the plasma membrane upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation and colocalizes with translocated p47phox and with p22phox, a subunit of flavocytochrome b558. Although activated Pak phosphorylated several essential serine residues in the C-terminus of p47phox, direct binding to p47phox was not observed. In contrast, active Pak bound directly to p22phox, suggesting flavocytochrome b was the oxidase-associated membrane target of this kinase and this association may facilitate further phosphorylation of p47phox in the assembling NADPH oxidase complex.


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