Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304.
The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.
Inanami
O
O
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Johnson
J L
JL
McAdara
J K
JK
Benna
J E
JE
Faust
L R
LR
Newburger
P E
PE
Babior
B M
BM
D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000097:blood*; D010750:Phosphoproteins / Q000097:blood* / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D017931:DNA Primers; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013481:Superoxides; D010768:Phosphoserine; D012694:Serine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013755:Tetradecanoylphorbol Acetate