title: Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity.
abstract: The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O(-)(2) at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47(phox) in vitro. Phosphoamino acid analysis of phosphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47(phox). Dependence of phosphorylation on the conformation of p47(phox) is supported by the finding that p47(phox) undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47(phox). In addition, 5,6-dichloro-1-beta-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase.
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
authors.forename: H S
authors.initials: HS
authors.lastname: Park
authors.forename: S M
authors.initials: SM
authors.lastname: Lee
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: Y S
authors.initials: YS
authors.lastname: Kim
authors.forename: Y S
authors.initials: YS
authors.lastname: Bae
authors.forename: J W
authors.initials: JW
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000494:pharmacology; D047390:Casein Kinase II; D002454:Cell Differentiation; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D018922:HL-60 Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000097:blood* / Q000737:chemistry; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000737:chemistry; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000097:blood* / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010750:Phosphoproteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D013481:Superoxides; D016718:Arachidonic Acid; D004004:Dichlororibofuranosylbenzimidazole; D009240:N-Formylmethionine Leucyl-Phenylalanine; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D004121:Dimethyl Sulfoxide