Phosphorylation of the leucocyte NADPH oxidase subunit p47phox 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.

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

“…production is associated with both the upstream/downstream kinase-dependent phosphorylation, and with the translocation of p47 phox and p67 phox to the plasma membrane (6). p47 phox phosphorylation in response to fMLP stimulation has been shown to be directly triggered by certain PKC isoforms, MAPK and Akt/PKB (32,(45)(46)(47)(48). Among the MAPKs, ERK1/2 have been shown to mediate the fMLP-stimulated phosphorylation of p47 phox , whereas inhibition of p38 MAPK had little impact on p47 phox phosphorylation (32,33).…”

p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases

“…Proinflammatory cytokines such as GM-CSF and TNFα which do not activate NADPH oxidase but prime its activation in response to a secondary stimulus such as fMLF (El Benna et al, 2008), induce partial phosphorylation of p47phox on Ser345 by ERK1/2 or p38MAPK and promote NADPH oxidase assembly (Dang et al, 1999(Dang et al, , 2006Dewas et al, 2003). While phosphorylation of p47phox by PKC, PAK and AKT in neutrophils has a positive stimulatory effect on NADPH oxidase activation and pre-phosphorylation by p38MAPkinase and ERK1/2 results in the enhancement of this effect, some data have suggested that the phosphorylation of p47phox by PKA or CKII could have a negative inhibitory effect (Bengis-Garber et al, 1996;Park et al, 2001).…”

“…In in vitro conditions, p47phox is phosphorylated on selective sites by different type of protein kinases such as PKC α, β, δ, and ζ (Dang et al, 2001a;Fontayne et al, 2002), PKA (El Benna et al, 1996a;Kramer et al, 1988), MAPKinase ERK2 and p38MAPKinase (El Benna et al, 1996b), protein casein kinase 2 (CKII) (Park et al, 2001), AKT (Chen et al, 2003;Hoyal et al, 2003), IRAK-4 (Pacquelet et al, 2007), p21-activated kinase (PAK) (Martyn et al, 2005), a phosphatidic acid-activated kinase (Waite et al, 1997) and src kinase (Chowdhury et al, 2005).…”

Regulation of Taurine Transport Systems by Protein Kinase CK2 in Mammalian Cells

“…This indicates that tyrosine phosphorylation is downstream in the swelling-induced taurine release signalling cascade and that ROS inhibit a protein tyrosine phosphatase, hence amplifying the effect of the protein tyrosine kinase. Several tyrosine kinases have been assigned a role in the regulation of the swelling-induced taurine release as judged from the effect of pharmacological inhibition (see [90,107] [108], possibly preventing interaction of the regulatory complex p40-p67-p47 phox with flavocytochrome b 558 , and thereby generation of ROS [108]. Activation of NOX can be induced by arachidonic acid which induces a conformational change in p47 phox , thereby activating the oxidase [109].…”