Published online before print October 2, 2009
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-PKC
,1
* Department of Physiology and Lung Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA;
Department of Pediatrics, University of Pennsylvania School of Medicine and the Children’s Hospital Research Institute, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; and
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
1. Correspondence: Department of Physiology and Lung Center, Temple University School of Medicine, 3307 North Broad St., PAH-206, Philadelphia, PA 19140, USA. E-mail: laurie.kilpatrick{at}temple.edu
ABSTRACT
In human neutrophils, TNF-elicited O2– production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O2– generation has yet to be fully elucidated. Previously, we identified 
-PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of -PKC with a dominant-negative -PKC TAT peptide resulted in a significant delay in the onset time of TNF-elicited O2– generation but had no effect on Vmax, indicating an involvement of -PKC in the initiation of O2– production. In contrast, fMLP-elicited O2– production in adherent and nonadherent neutrophils was -PKC-independent, suggesting differential regulation of O2– production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time-dependent association of -PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for -PKC in regulating O2– production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O2– generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was -PKC-dependent. -PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of -PKC (Ser643) and -PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus, signaling for TNF-elicited O2– generation is regulated by -PKC. Adherence-dependent cooperative signaling activates PI3K signaling, -PKC phosphorylation, and -PKC recruitment to p47phox. -PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.
Key Words: superoxide anion generation • p47phox • fMet-Leu-Phe • ERK • PI 3-kinase • phosphorylation
