Phosphorylation of the Respiratory Burst Oxidase Subunit p67 during Human Neutrophil Activation

Abstract

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O 2 . ) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67 phox. In this study, we found that activation of human neutrophils with formyl-methionylleucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67 phox . Using an anti-p67 phox antibody or an antip47 phox antibody, we showed that phosphorylated p67 phox and p47 phox form a complex. Phosphoamino acid analysis of the phosphorylated p67 phox revealed only 32 P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67 phox is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67 phox in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67 phox was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67 phox is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…The lack of effects on gp91 phox gene expression suggests that cascade overactivation can antagonize functions of NADPH oxidase components via post-translational modifications. Indeed, serine phosphorylation regulates the activities of several components, including p47 phox , p67 phox and Rap1, and threonine phosphorylation of p40 phox inhibits NADPH oxidase function [45][46][47][48]. Despite this antagonistic role, the cascade is required in mature neutrophils but CKLiK expression levels may be insufficient to positively regulate NADPH oxidase components in the absence of CaMKKα activities.…”

Reconstitution of Chemotactic Peptide-Induced Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activation in Transgenic COS-phox Cells

“…generation (29). PKC-regulated phosphorylation of p47 phox is also observed in fMLP-induced NADPH oxidase activation (32)(33)(34)(35), but the responsible PKC isoforms have not been identified. A major observation of the current study is that PKC␦ plays a key role in FPR-mediated NADPH oxidase activation in both transgenic COS-phox cells and human neutrophils.…”

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox

“…Normal human neutrophils and neutrophils from two p67 phox -deficient CGD patients were obtained by Dextran sedimentation and Ficoll centrifugation [10]. The cells (50 ϫ 10 6 /mL) were suspended in phosphate-buffered saline (PBS) and treated with 2.7 mM DFP for 15 min at 4°C, then washed and resuspended in Hanks' buffer containing Ca 2ϩ , Mg 2ϩ , and D-glucose.…”

“…After 30 min of incubation on ice the fraction was centrifuged at 100,000 g for 30 min at 4°C, and protein was immunoprecipitated [8,10].…”

“…In resting cells p67 phox , p47 phox , and p21rac are located in the cytosol. On stimulation, p47 phox and p67 phox undergo phosphorylation [6][7][8][9][10] and translocate to the plasma membrane [11][12][13][14]. Biochemical studies suggest that the cytosolic components form a complex of about 240 kDa [15].…”