Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by caseinkinase 2: conformation-dependent phosphorylation and modulation of oxidaseactivity
Hs. Park et al., Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by caseinkinase 2: conformation-dependent phosphorylation and modulation of oxidaseactivity, BIOCHEM J, 358, 2001, pp. 783-790
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 en
zyme is dormant in resting neutrophils but becomes active when the cells ar
e exposed to the appropriate stimuli. During oxidase activation, the highly
basic cytosolic oxidase component p47(phox) becomes phosphorylated on seve
ral serines and migrates to the plasma membrane. Protein kinase CK2 is an e
ssential serine/threonine kinase present in all eukaryotic organisms. The l
eucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphory
lation sites. In the present study, we report that CK2 is able to catalyse
the phosphorylation of p47(phox) in vitro. Phosphoamino acid analysis of ph
osphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on
serine residues. CNBr mapping and phosphorylation of peptides containing th
e 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 confo
rmation of p47(phox) is supported by the finding that p47(phox) undergoes b
etter phosphorylation by CK2 in the presence of arachidonic acid, a known a
ctivator of NADPH oxidase which induces conformational changes in p47(phox)
. In addition, 5,6-dichloro-1-beta -o-ribofuranosyl benzimidazole, a CK2 in
hibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in D
MSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the
p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates th
e deactivation of NADPH oxidase.