Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils

Abstract

p47(phox) is an essential component of the NADPH oxidase, and phosphorylation of p47(phox) is associated with activation of the enzyme. Here we have used p47(phox) affinity chromatography to extract a p47(phox) kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-beta(I), -beta(II) and -delta. The C-terminus of p47(phox) represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47(phox), we investigated the functional relevance of the interaction between PKC and p47(phox). Subcellular fractionation revealed an abnormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to particulate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47(phox) affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47(phox) can act as a regulator of PKC in neutrophils.

Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid

“…PKC in PMN-PMN express ␣, ␤ I , ␤ II , ␦, and but not several other PKC isoforms (25,29,30,(43)(44)(45)(46)(47)(48)(49). We confirmed that PMN contain the former but lack ⑀, , , or isoforms (36,37).…”

Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst

“…Unsaturated fatty acids are well-established modulators of protein kinase C activity (McPhail et al 1984;O'Flaherty and Nishihira 1987;Shinomura et al 1991). PMA-induced respiratory burst activation requires protein kinase C and this activation step has been implicated in p47phox phosphorylation (Allard et al 1999;Reeves et al 1999;Korchak et al 1998). However, PMA-induced phosphorylation of p47phox is observed irrespective of cotreatment with methyl 9,10-/12,13-DiHOME ( figure 8).…”

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

“…Translocation of p47 phox to the membrane requires phosphorylation [55] of a number of serine sites located in its C-terminus, resulting in exposure of its SH3 domain, which binds directly to the p22 phox subunit of the flavocytochrome [56]. Neutrophils contain five of the eleven isoforms of PKC and there are several lines of evidence supporting a role for protein kinase C (PKC) a, bII, d and z in the phosphorylation of p47 phox [57] with a direct interaction between this substrate and the kinase been observed [58]. PKC is also capable of phosphorylating p47 phox at various sites, which are absolutely essential for activation and translocation of the protein [59].…”