Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47(phox) and p67(phox) subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47(phox) and p67(phox) are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47(phox) homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67(phox) homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47(phox) was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).
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PMID:Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. 1247 64

gp91(phox) (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47(phox) and p67(phox). Nox1, a homolog of gp91(phox), is regulated by NOXO1 and NOXA1, homologs of p47(phox) and p67(phox), respectively. For both Nox1 and gp91(phox), an organizer protein (NOXO1 or p47(phox)) cooperates with an activator protein (NOXA1 or p67(phox)) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91(phox), was activated by p47(phox) plus p67(phox). Whereas gp91(phox) activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated approximately 30% by PMA. gp91(phox) was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91(phox) regulation required an intact activation domain in the activator protein, as neither p67(phox)(V204A) nor NOXA1(V205A) were effective. In contrast, p67(phox)(V204A) was effective (along with p47(phox)) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67(phox). Nox3 activity was regulated by PMA only when p47(phox) but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47(phox) but not in NOXO1. Deletion of the autoinhibitory region from p47(phox) rendered this subunit highly active in the absence of PMA toward both gp91(phox) and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.
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PMID:Nox3 regulation by NOXO1, p47phox, and p67phox. 1518 Oct 5

The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.
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PMID:Mechanism of angiotensin II-induced superoxide production in cells reconstituted with angiotensin type 1 receptor and the components of NADPH oxidase. 1798 2

NADPH oxidase activator 1 (NOXA1) together with NADPH oxidase organizer 1 (NOXO1) are key regulatory subunits of the NADPH oxidase NOX1. NOX1 is expressed mainly in colon epithelial cells and could be involved in mucosal innate immunity by producing reactive oxygen species (ROS). Contrary to its phagocyte counterpart NOX2, the mechanisms involved in NOX1 activation and regulation remain unclear. Here we report that NOX1 activity is regulated through MAP kinase (MAPK), protein kinase C (PKC), and protein kinase A (PKA)-dependent phosphorylation of NOXA1. We identified Ser-282 as target of MAPK and Ser-172 as target of PKC and PKA in vitro and in a transfected human embryonic kidney 293 (HEK293) cell model using site directed mutagenesis and phosphopeptide mapping analysis. In HEK293 cells, phosphorylation of these sites occurred at a basal level and down-regulated constitutive NOX1 activity. Indeed, S172A and S282A single mutants of NOXA1 significantly up-regulated constitutive NOX1-derived ROS production, and S172A/S282A double mutant further increased it, as compared to wild-type NOXA1. Furthermore, phosphorylation of NOXA1 on Ser-282 and Ser-172 decreased its binding to NOX1 and Rac1. These results demonstrated a critical role of NOXA1 phosphorylation on Ser-282 and Ser-172 in preventing NOX1 hyperactivation through the decrease of NOXA1 interaction to NOX1 and Rac1.
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PMID:Phosphorylation of NADPH oxidase activator 1 (NOXA1) on serine 282 by MAP kinases and on serine 172 by protein kinase C and protein kinase A prevents NOX1 hyperactivation. 2011 Feb 67

Reactive oxygen species (ROS) production by NADPH oxidase 1 (NOX1), which is mainly expressed in colon epithelial cells, requires the membrane-bound component p22(PHOX) and the cytosolic partners NOX organizer 1 (NOXO1), NOX activator 1 (NOXA1), and Rac1. Contrary to that of its phagocyte counterpart NOX2, the molecular basis of NOX1 regulation is not clear. Because NOXO1 lacks the phosphorylated region found in its homolog p47(PHOX), the current view is that NOX1 activation occurs without NOXO1 phosphorylation. Here, however, we demonstrate that phorbol myristate acetate (PMA) stimulates NOXO1 phosphorylation in a transfected human embryonic kidney (HEK) 293 epithelial cell model via protein kinase C and identify Ser-154 as the major phosphorylated site. Endogenous NOXO1 from T84 colon epithelial cells was also phosphorylated, suggesting that NOXO1 phosphorylation is physiologically relevant. In transfected HEK-293 cells, PMA-induced phosphorylation on Ser-154 enhanced NOXO1 binding to NOXA1 (+97%) and to the p22(PHOX) C-terminal region (+384%), increased NOXO1 colocalization with p22(PHOX), and allowed optimal ROS production by NOX1 as demonstrated by the use of S154A and S154D mutants compared with that by wild-type NOXO1 (P<0.05). Pulldown experiments revealed that phos-phorylation on Ser-154 was sufficient to markedly enhance NOXO1 binding to NOXA1, which in turn acts as a molecular switch, allowing optimal interaction of NOXO1 with p22(PHOX). This study unexpectedly revealed that full assembly and activation of NOX1 is a tightly regulated process in which NOXO1 phosphorylation on Ser-154 is the initial trigger.
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PMID:NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation. 2332 65