Novel Mechanism of Activation of NADPH Oxidase 5

Abstract

In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC50 of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr494 and Ser498. Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr494 and Ser498 to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli. In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC50 of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr494 and Ser498. Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr494 and Ser498 to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli. The NADPH oxidases or Noxs define a unique family of enzymes that exist to synthesize reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; RLU, relative light units; PMA, phorbol 12-myristate 13-acetate; WT, wild type; GFP, green fluorescent protein; RFP, red fluorescent protein; EGFP, enhanced green fluorescent protein; PKC, protein kinase C; MOPS, 4-morpholinepropanesulfonic acid; CIP, calf intestinal phosphatase; DPI, diphenyleneiodonium; SOD, superoxide dismutase. (1Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2468) Google Scholar). Currently, five distinct Noxs have been identified and are numbered Nox1 to -5. Of these, Nox2 is the best characterized and is expressed primarily by cells of the immune system, including neutrophils and macrophages. Mutation of Nox2 or targeted disruption in mice results in impaired host defense (2Babior B.M. Curr. Opin. Immunol. 2004; 16: 42-47Crossref PubMed Scopus (655) Google Scholar). The biological functions of Noxs that are expressed in cells outside of the immune system are emerging but remain somewhat enigmatic. However, the considerable diversity that exists in the location, the capacity, and the mechanisms by which these enzymes are activated suggests that they have indeed evolved distinct functional roles. The mechanisms that control the synthesis of ROS by the various Nox isoforms are remarkable in their complexity. Nox2 is a transmembrane glycoprotein that is activated by the coordinated assembly of at least five distinct subunits. In unstimulated cells, Nox2 is bound to the protein p22phox, and upon activation, the coalescence of p67phox, p47phox, p40phox, and Rac yields a functional, superoxide-generating oxidase (1Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2468) Google Scholar, 3Sumimoto H. Miyano K. Takeya R. Biochem. Biophys. Res. Commun. 2005; 338: 677-686Crossref PubMed Scopus (256) Google Scholar). Nox1 is also bound to p22phox and is activated by the subunits NOXA1 and NOXO1, which are functionally related to p67phox and p47phox, respectively (4Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 5Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). The activity of Nox3 can be regulated by p22phox, p67phox, p47phox/p67phox, or NOXO1, but compared with Nox2, it is less discriminating in which subunits are required for full activation (6Cheng G. Ritsick D. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 7Banfi B. Malgrange B. Knisz J. Steger K. Dubois-Dauphin M. Krause K.H. J. Biol. Chem. 2004; 279: 46065-46072Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The activity of Nox4 is largely constitutive, but it is at least in part dependent on the co-association of p22phox for full activity (8Kawahara T. Ritsick D. Cheng G. Lambeth J.D. J. Biol. Chem. 2005; 280: 31859-31869Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 9Ambasta R.K. Kumar P. Griendling K.K. Schmidt H.H. Busse R. Brandes R.P. J. Biol. Chem. 2004; 279: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). In contrast, Nox5 is a calcium-dependent enzyme and does not depend on the assembly of other proteins, including p22phox, for its activity (8Kawahara T. Ritsick D. Cheng G. Lambeth J.D. J. Biol. Chem. 2005; 280: 31859-31869Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 10Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Nox5, like the other Nox isoforms, is a membrane-bound oxidase that spans the membrane six times (10Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). However, Nox5 is distinct in that its N terminus contains four EF hands that bind calcium and regulate its activity. In a cell-free system, the activity of membrane-bound Nox5 was shown to be entirely dependent on calcium. The addition of cytosol or cytosolic proteins that can activate Nox2 in cell-free assays did not increase the activity of Nox5 (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The amino terminus of Nox5 operates in a manner analogous to calmodulin, whereby calcium binding initiates a conformational change in the enzyme. This permits the interaction of the N terminus of Nox5 with a yet to be identified C-terminal domain, which then facilitates electron delivery and the production of reactive oxygen species (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In cell-free assays, the EC50 for calcium was determined to be ∼1 μm, which is relatively high and unlikely to be achieved in most cells outside of extraordinary circumstances (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In the context of the above model of Nox5 activation, the current study identifies a novel mechanism regulating the activity of Nox5 and provides a more physiological context for enzyme activation at lower levels of intracellular calcium. Cell Culture and Transfection—COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing l-glutamine, penicillin, streptomycin, and 10% (v/v) fetal bovine serum. Cells were transfected with Lipofectamine™ 2000 according to the manufacturer’s instructions (Invitrogen). Measurement of Reactive Oxygen Species—COS-7 cells were transfected with cDNAs encoding Nox5 or control plasmids (GFP, RFP, or LacZ), and 24 h later, cells were replated into white tissue culture-treated 96-well plates (ThermoLabsystems) at a density of ∼5 × 104 cells/well. The cells were incubated at 37 °C in phenol-free Dulbecco’s modified Eagle’s medium (Sigma) containing a 400 μm concentration of the luminol analogue L-012 (Wako) for a minimum of 20 min prior to the addition of agonists (12Daiber A. August M. Baldus S. Wendt M. Oelze M. Sydow K. Kleschyov A.L. Munzel T. Free Radic. Biol. Med. 2004; 36: 101-111Crossref PubMed Scopus (149) Google Scholar). Luminescence was quantified over time using a Lumistar Galaxy (BMG) luminometer. The specificity of L-012 for reactive oxygen species was confirmed by transfecting cells with a control plasmid, such as GFP or LacZ or by co-incubation of a superoxide scavenger, such as TIRON (5 mm). Both of these interventions yielded virtually undetectable levels of luminescence under control or PMA- or ionomycin-stimulated conditions. the relative light quantified from the luminescence of L-012 are of in the activity of Nox5. In the activity of Nox5 was also using a that is for superoxide was into a modified containing an and was by the of the of and Nox5 were and into plasmids as D. D. J. 2005; PubMed Scopus Google Scholar). endoplasmic and were from and have been D. D. J. 2005; PubMed Scopus Google Scholar, D. J.D. D. Biol. PubMed Scopus Google Scholar). Nox5 encoding the and was by using Nox5 as a and the were with the and were with the of function mutant was by were confirmed by cells were incubated with a of and with μm and bovine for min and then incubated in the for min to of the was then with and cells were in a and to the was into intracellular by using the was the in the presence of and was the in the presence on and and was the for D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Cell cells were transfected with cDNAs encoding proteins of and S. A. PubMed Scopus Google as h later, cells were replated was using the of was at with was from were by as D. J. K. A. PubMed Scopus Google and with to which the amino acid and is and to Thr494 and Ser498 were by using the were by and The specificity of these was determined the Nox5 protein as shown in and relative of Nox5 was determined using the of cells transfected with Nox5 were to PMA or and into and according to the manufacturer’s In cells were with and then using a of four was then with and proteins to the the of Nox5, cells were in a and × on and by × for into and cells expressing Nox5 were in a containing and cells were and the was and then times with were in the above containing to calcium. were at low and at The was then and the was in with The cell-free was into containing μm and calcium from to μm calcium In of calf intestinal or were and incubated at °C for h prior to the addition of calcium. a of NADPH (Sigma) was to a concentration of μm, and the production of reactive oxygen species was over was a from are expressed as were using and were using a or of with a were as at of Nox5 by shown the activity of Nox5 can be regulated by intracellular calcium. In the addition of a rapid increase in the production of ROS compared with cells with In the presence of the and the superoxide and is that it from a Nox However, as shown in the addition of a slow and sustained production of ROS that is over The analogue of did not ROS DPI, SOD, and TIRON abolished PMA-dependent ROS Nox as the of ROS direct of cells expressing Nox5 a control GFP, that of the ROS produced in response to both PMA and from Nox5 of PMA Nox5 the the activation of Nox5 with PMA, cells expressing Nox5 were with PMA prior to the addition of different concentrations of ionomycin. shown in the addition of a relatively low concentration of a increase in Nox5 activity of × × The of PMA luminescence from × to × but PMA potentiated the low response to a of × × RLU, or in the of PMA concentrations of is a more increase in Nox5 activity of × × with PMA increased Nox5 activity to levels as shown in but the in of the PMA response was × × and not different from that in the of more was the ability of PMA to the of Nox5 activation as from the under the above In response to the low of the under the in the presence of PMA was greater compared with control cells the PMA as the of was less and greater in the of Nox5 by PMA and on and cells expressing Nox5 were with or acid and stimulated with or PMA shown in intracellular and extracellular calcium are both required for full activation of Nox5 in response to ionomycin. of intracellular calcium the activity of Nox5 and the of of extracellular calcium did not the of activation but activity. The of the activity. The response to PMA was since chelation of intracellular or extracellular calcium virtually abolished Nox5 activity the that PMA is intracellular calcium and Nox5, we the of intracellular calcium using the fluorescent intracellular calcium shown in PMA does not the of intracellular calcium or the increase in response to as shown by the of calcium over In the increase in intracellular calcium in response to lower concentrations of was not by PMA not whether PMA was a direct change in Nox5 activity, we activity assays in cell-free conditions. shown in Nox5 from cells an increase in calcium sensitivity compared with the activity of control and Nox5 was whether phosphorylation was for the change in calcium were with to Nox5. shown in the calcium sensitivity of Nox5, that phosphorylation is required for the calcium sensitization. The cell-free was for Nox5 activity, since was ROS production in cells expressing a control and also since ROS production in cells expressing Nox5 was by DPI, SOD, and TIRON The relative calcium-dependent activity in cells to PMA is shown in Nox5 in the and by cells expressing Nox5 were into and shown in Nox5 is primarily in the with a detected in the membrane The of was confirmed by the presence of in the in in the and in the that Nox5 was in the cells were in a and into and shown in virtually of the Nox5 was in the the detergent-resistant of Nox5 and its presence in the from using to Nox5 from other proteins and its phosphorylation we to Nox5 in the and its phosphorylation using an that upon with PMA, but not or Nox5 is in cells transfected with a control plasmid, GFP, was in at the as Nox5. phosphorylation with ROS we compared the activity of Nox5 with its phosphorylation Exposure of cells to PMA the time-dependent phosphorylation of Nox5, which was and min and the time-dependent increase in Nox5 activity. The of Nox5 on was to Nox5 phosphorylation for on the of and the a of phosphorylation was identified at and Ser498 of these phosphorylation abolished both phosphorylation and the ability of PMA to phosphorylation the functional of these phosphorylation cells were transfected with Nox5 and mutant Nox5 in which and Ser498 were to the shown in the ability of PMA to Nox5 activity was greatly in the mutant compared with on Nox5 activity, we the ability of high of to the calcium-dependent was in the ability of to ROS production from and mutant Nox5 This that the of the PMA-dependent phosphorylation ROS in response to PMA but does not Nox5 activity Mutation of PMA-dependent cells were with PMA or and then stimulated with different concentrations of ionomycin. concentrations of a increase in ROS production that was greatly potentiated by with PMA This was greatly in cells expressing the Nox5 the response to was to that of the Nox5 in the of PMA also the of increased Nox5 activity as in and B. the concentration of increased the effect of PMA the was that the of Nox5 to ROS was calcium. and of Nox5 the we of the phosphorylation and compared the activity to shown in the activity of the mutant was to that of WT, but mutation of and Ser498 produced from to whether more phosphorylation was required for full activity. In and cells were transfected with and and and with produced greater the with the the most The mutant also produced of activity to the that these have on the activity of Nox5, we the response to high concentrations of ionomycin. shown in and the response to was not compared with of more phosphorylation is required for full Nox5 activity in response to cells expressing WT, and Nox5 were stimulated with PMA relative activity of WT, and Nox5 in the presence of μm, cells expressing and Nox5 were stimulated with PMA relative activity of and Nox5 in the presence of μm, cells expressing and Nox5 were stimulated with PMA relative activity of and Nox5 in the presence of μm, the phosphorylation on Nox5 by the we with containing WT, or Nox5. shown in the the Ser498 as by the of with the the of these we phosphorylation to Thr494 and Ser498. In we that PMA the phosphorylation of Ser498. The specificity of is by the with and in the presence of the alanine the phosphorylation of Thr494 is increased in response to PMA and abolished by or mutation the functional of these we Thr494 and Ser498 to the glutamic cells expressing the mutant produced more ROS in response to low concentrations of the presence of of protein and of on Nox5 whether PMA-dependent activation of Nox5 PKC, cells expressing Nox5 were incubated with the and Both and PMA-dependent in Nox5 activity also the response to that its may not be entirely for the phosphorylation of Ser498 and to a results that or a in the phosphorylation and activation of Nox5. of the of the of Nox5 cells, we GFP and Nox5 these proteins were and produced ROS in response to both and PMA not In transfected cells, a with an of that into the cytosol in Nox5 did not with for the or the and Nox5 was also from the and the membrane and However, considerable was detected with the for the endoplasmic we had detected Nox5 in the using that it does not with for the including both and and The variety and of the mechanisms the activity of NADPH oxidases is Nox1 to require the presence of accessory proteins, p22phox, p47phox, p40phox, p67phox, NOXO1, and in various to activate superoxide production H. Miyano K. Takeya R. Biochem. Biophys. Res. Commun. 2005; 338: 677-686Crossref PubMed Scopus (256) Google Scholar). Nox5 is different Currently, it that Nox5 can of of the accessory proteins for the other Noxs (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). the activity of Nox5 is by the elevation of intracellular calcium. In the production of superoxide from Nox5 is compared with the activation to by the other However, the of intracellular calcium required for activation of Nox5 is relatively high at μm for and activation, which raises the question of whether cells experience calcium for Nox5 activation (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In the current we have identified a novel mechanism that the activity of Nox5 by its sensitivity to lower levels of intracellular calcium and by the of superoxide PMA of cells expressing Nox5 resulted in a slow and production of ROS above This from the of the response to ionomycin. The of PMA and results in a of both the and of ROS at low concentrations of ionomycin. The ability of PMA to the response with concentration of and was dependent on since chelation of both and extracellular calcium abolished This ability of PMA to Nox5 activity was a direct effect on the since prior of cells to PMA Nox5 to calcium in cell-free activity This calcium was by with that direct phosphorylation of Nox5 PMA did not the concentration of intracellular under or in the presence of ionomycin. these results the that PMA the calcium sensitivity of Nox5. In response to PMA, we also detected the time-dependent phosphorylation of Nox5. Mutation of and residues and to the both the the PMA-dependent increase in ROS production and the ability of PMA to ROS production at low concentrations of ionomycin. the that these Nox activity in response to we that in the presence of concentrations of the activity of the Nox5 mutant was to that of in the presence of calcium Nox5 is dependent on the phosphorylation of these for full activity. mutation of these phosphorylation produced that they may function In of or and resulted in less PMA-dependent activity. Mutation of to the glutamic acid produced a gain of function mutant that greater activity in response to lower concentrations of and provides that the phosphorylation of Nox5 activity. the of we phosphorylation to Thr494 and Ser498 and characterized the specificity of the using that the Ser498. using the to Thr494 and we that PMA the phosphorylation of Thr494 and Ser498. The kinase for the phosphorylation of Nox5 is not yet However, both phosphorylation and PMA-dependent in activity were to the that or a The mechanisms by which phosphorylation of Nox5 facilitates the calcium-dependent synthesis of ROS are it been shown that the EF hands of Nox5 with its terminus to ROS The binding of calcium to the EF hands of Nox5 a conformational change that a This then with a yet to be identified C-terminal domain, to increased electron and ROS production (11Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In the presence of PMA, both the and of the calcium-dependent Nox5 activity are is that phosphorylation facilitates or the interaction these protein and that more phosphorylation is required to in the Thr494 and Ser498 phosphorylation outside of the six transmembrane and are to be and to of transmembrane a transmembrane that the and NADPH binding The of is not and are related enzymes that also EF hands and ROS in a calcium-dependent manner (1Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2468) Google Scholar, R. S. D. D. S. R. J. K. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). of the amino acid of Nox5 and and that the containing the Nox5 phosphorylation is in and The functional of is not yet of the amino acid of the more related Nox2 and Nox4 with Nox5 also that is unique to Nox5. on the results of the cell-free activity assays, it is that the phosphorylation of is to increase the sensitivity of Nox5 to calcium. However, it is not yet whether the ability of Nox5 to bound calcium in an of calcium concentration and the enzyme in an activated or to increase the of the enzyme for calcium. the current is the that Nox5 can be activated by Previous studies have been to that PMA Nox5 activity in However, these studies were since it was not whether the cells in question Nox5 superoxide production was and is that the cells had the to to S. J. PubMed Scopus Google Scholar). have shown that PMA does not regulate the of Nox5 in cells of and that superoxide production is by of protein kinase with K. Krause K.H. M. J. Immunol. 2005; PubMed Scopus Google Scholar). The kinase that Nox5 in response to PMA in the current study is not yet studies that Nox5 is a protein that with However, may not its intracellular location, and we GFP and proteins and the of Nox5 in cells using studies that Nox5 is with an of that into the This is a of a protein that in the endoplasmic M. F. S. N. J. J. Cell Biol. 2003; PubMed Scopus Google and indeed was considerable with the endoplasmic but not for other proteins, such as the or In Nox5 was from the and the The of Nox5 is to that for Nox2 and Nox4 J.D. M. 2005; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). The functional of Nox5 to microdomains of the endoplasmic is not we have shown that PMA facilitates the calcium dependent activation of Nox5. This phosphorylation of residues and and enhanced enzyme activation at lower and more concentrations of intracellular calcium.