Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA–Nox2 fusion proteins to p67phox. We found, serendipitously, that deletion of residues 259–279 in p67phox (p67phoxΔ(259–279)), endowed it with the ability to bind selectively to Nox2 peptide 369–383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375–383. Binding of p67phoxΔ(259–279) to peptide 28 was mimicked by that of a (p67phox‐RacGTP) chimera. Both p67phoxΔ(259–279) and the (p67pho–RacGTP) chimera bound a NusA–Nox2 fusion protein, comprising residues 375–383. Specific single residue deletion mutants, within the p67phox sequence 259–279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259–279 sequence in p67phox, were found to autobind p67phox, suggesting that an intramolecular bond exists in p67phox, one pole of which was located within residues 259–279. We conclude that “resting” p67phox exists in a “closed” conformation, generated by an intramolecular bond. Deletion of specific residues within the 259–279 sequence, in vitro, or interaction with RacGTP, in vivo, causes “opening” of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

The superoxide (O·−2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O·−2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus. We demonstrate that the new PKC-related family member, PKC-L, recently isolated by us, is expressed specifically in the cell nucleus. Localization of PKC-L in the cell nucleus is shown both by immunofluorescence staining and by subcellular fractionation experiments of several human vell lines, including the human epidermoid carcinoma line A431. Treatment of these cells by phorbol esters does not induce the down-regulation of PKC-L, in contrast to their effect on classical PKC family members. This is the only PKC isoenzyme described so far that resides permanently and specifically in the cell nucleus. PKC-L may function as an important link in tumor promoting, e.g., as a nuclear regulator of gene expression that changes the phosphorylation state of transcriptional components such as the AP-1 complex.Protein kinase C (PKC) is involved in one of the major signal transduction systems, activated upon external stimulation of cells by various ligands, including hormones neurotransmitters and growth factors. These external stimuli increase the level of sn-1,2-diacylglycerol, which functions as a second messenger by binding and activating PKC (6,27,28). Phorbol esters and other tumor promoters are potent activators of PKC by mimicking the effects of its natural activator, diacylglycerol (27). Activation of PKC, the highaffinity phorbol ester receptor (4,22,26), is regarded as responsible, at least in part, for their tumor promotion activity.Molecular cloning has revealed that PKC exists as a family of multiple subspecies having closely related structures.Four subspecies, a, PI, I and y, were initially isolated (11,16,19,30,33,35). Later, five additional cDNA clones, designated E, 8, C, -9, and L, were characterized (5,29,31,32,34). These latter clones, also termed PKC-related family members, have structural features clearly distinct from those of the four subspecies initially isolated. Most notably, they lack the conserved (C2) region, presumably involved in Ca2+ binding (5,29,31,32,34). The human PKC-L gene, recently isolated by us (5), and its murine homolog, nPKC-(34), have a unique tissue distribution. They were shown to be expressed predominantly in lung, heart, and skin, while the expression of all other mammalian members of the PKC gene family is enriched in brain tissues (5,34).Emerging data on the structure, biochemical properties, and tissue distribution of the individual PKC family members have suggested that these are not mere isoenzymes of identical function but rather that different enzymes may execute distinct cellular functions (27, 28). The fact that more than one isoform is usually expressed within a particular cell type (3,17,40) Plasmid pXKF, coding for PKC-a...

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