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Biochem Pharmacol. 2011 Nov 1;82(9):1145-52. doi: 10.1016/j.bcp.2011.07.070. Epub 2011 Jul 20.

The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A.

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INSERM, U773, Centre de Recherche Biom├ędicale Bichat Beaujon CRB3, Paris F-75018, France.


Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.

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