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Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for α-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of β-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase. Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for α-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of β-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase. There is now substantial evidence that oxidant species such as H2O2 are produced in a regulated way in cells where they can function as signaling agents (1Rhee S. G. Bae Y. -S. Lee S. -R. Kwon J. Science's STKE. 2000www. stke. org/cgi/content/full/OCₛigtrans;2000/53/pe1Google Scholar, 2Reth M. Nat. Immunol. 2002; 3: 1129-1134Crossref PubMed Scopus (611) Google Scholar). We have been studying the post-translational modification of protein cysteinyl thiols, as this is a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. Our strategy has been to search for proteins that are susceptible to a variety of different modes of cysteine oxidation, such as S-thiolation (3Eaton P. Byers H. L. Leeds N. Ward M. A. Shattock M. J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 4Eaton P. Jones M. E. McGregor E. Dunn M. J. Leeds N. Byers H. L. Leung K. Y. Ward M. A. Pratt J. Shattock M. J. J. Am. Soc. Nephrol. 2003; 14: S290-S296Crossref PubMed Google Scholar), sulfenation (5Saurin A. T. Neubert H. Brennan J. P. Eaton P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17982-17987Crossref PubMed Scopus (243) Google Scholar), and protein-protein disulfide bond formation (6Brennan J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The rationale is that once we identify proteins with reactive thiols, the possibility that their oxidation has a functional correlate of physiological significance can be investigated. We previously found the RI regulatory subunits of protein kinase A (PKA) 2The abbreviations used are: PKA, protein kinase A; AKAP, protein kinase A anchor protein; ARVM, adult rat ventricular myocytes; PBS-T, phosphate-buffered saline plus 1% Triton X-100; α-MyHC, α-myosin heavy chain. 2The abbreviations used are: PKA, protein kinase A; AKAP, protein kinase A anchor protein; ARVM, adult rat ventricular myocytes; PBS-T, phosphate-buffered saline plus 1% Triton X-100; α-MyHC, α-myosin heavy chain. form interprotein disulfide dimers during cardiac oxidative stress (6Brennan J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Here we investigated the potential impact of this disulfide dimer formation on the function of PKA. PKA has two major forms (type I and type II), both of which exist as a tetramer comprising two catalytic and two regulatory subunits. There are two types of regulatory subunits (RI and RII), the presence of which in the PKA holokinase nominally defines the enzyme as type I or II, respectively. Recent studies have shown that the full dissociation of type I PKA in response to cAMP requires the presence of a substrate (7Vigil D. Blumenthal D. K. Brown S. Taylor S. S. Trewhella J. Biochemistry. 2004; 43: 5629-5636Crossref PubMed Scopus (50) Google Scholar). This substrate-induced sensitization of type I PKA is not a feature of the type II enzyme (8Viste K. Kopperud R. K. Christensen A. E. Doskeland S. O. J. Biol. Chem. 2005; 280: 13279-13284Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The regulatory subunits contain N-terminal sequences that are important for protein kinase A anchor protein (AKAP) binding. AKAPs are a diverse group of proteins that are found next to PKA substrate proteins and, thus, function to target PKA (9Michel J. J. Scott J. D. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 235-257Crossref PubMed Scopus (282) Google Scholar). Type I PKA is located in the cytosol, whereas type II is not as a result of being primarily bound (targeted) to AKAP proteins that are associated with various subcellular compartments, including the myofilaments in myocytes. RI and RII have significant homology, but one notable difference is the presence of a pair of N-terminal cysteine residues in RI. These cysteines have been thought to form constitutively present interprotein disulfides between RI subunits (10Banky P. Huang L. J. Taylor S. S. J. Biol. Chem. 1998; 273: 35048-35055Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), which align anti-parallel to each other and form disulfide bonds linking Cys-17 and -38 (in rat) of different RI molecules (11Leon D. A. Herberg F. W. Banky P. Taylor S. S. J. Biol. Chem. 1997; 272: 28431-28437Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, here we report RI does not, in fact, exist constitutively as a disulfide dimer. The RI disulfide dimer only forms when prooxidizing conditions exist. In cardiac tissue this redox change induces a subcellular translocation and kinase activation, resulting in phosphorylation of multiple PKA substrates, which increases the amplitude of myocyte contraction. The disulfide formation and PKA activation are causatively linked, as a selective inhibitor of this kinase (H89) prevents these changes. This oxidant-induced modification and activation of PKA as well as phosphorylation of established PKA substrates occurs without elevations in cAMP, further supporting a functional consequence of the oxidative structural modification. Animals—Animals received humane care in compliance with the “Principles of Laboratory Animal Care” as published by the National Institutes of Health (NIH Publication no. 85-23). Rats were anesthetized with sodium pentobarbitone (100 mg kg−1 intraperitoneal). Fresh and Cultured Rat Myocyte Preparation—Calcium-tolerant adult rat ventricular myocytes (ARVM) were isolated from male Wistar rats (250–300 g) as before (12James A. F. Ramsey J. E. Reynolds A. M. Hendry B. M. Shattock M. J. Biochem. Biophys. Res. Commun. 2001; 284: 1048-1055Crossref PubMed Scopus (27) Google Scholar) and kept at room temperature in modified Tyrode buffer for 3 h or cultured overnight as described previously (13Snabaitis A. K. Muntendorf A. Wieland T. Avkiran M. Cell. Signal. 2005; 17: 655-664Crossref PubMed Scopus (58) Google Scholar). Fresh ARVM were treated with H2O2 (1 μm–10 mm) (Sigma) for 5 min. Cells were centrifuged at 500 × g for 30s and then reconstituted in nonreducing SDS sample buffer containing maleimide (100 mm). In a separate experiments ARVM were treated with H2O2 (100 μm) or isoprenaline (100 nm) (Sigma) for 5 min with or without a 5-min pretreatment of H89 (10 μm) (Calbiochem) and again prepared in maleimide sample buffer. In cultured ARVM, after 18 h of incubation H2O2 (1 μm–1 mm) was added to the culture medium for 5 min. The culture medium was then aspirated, and the myocytes were scraped into 100 μl of maleimide sample buffer. Crystalloid Perfusion of the Rat Heart—Hearts from male Wistar rats were prepared and buffer-perfused as before (3Eaton P. Byers H. L. Leeds N. Ward M. A. Shattock M. J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). After 25 min of aerobic perfusion, hearts were perfused at a constant flow with Krebs-Henseleit bicarbonate buffer containing H2O2 (1 μm–10 mm) or isoprenaline (100 nm) for 5 min or with H2O2 (100 μm) for 1–10 min. Ventricles were snap-frozen and homogenized (10 ml buffer/g tissue) on ice in 100 mm Tris-HCl, pH 7. 2, plus maleimide (100 mm) and protease inhibitors (Complete C, Roche Applied Science) using a tissue was reconstituted in maleimide SDS sample buffer. subcellular the was centrifuged at × g for 5 and the was as The was in buffer containing 1% Triton and centrifuged as with the in proteins and the in myofilament and proteins. was using the After were to using a in This used in these studies PKA RI and PKA substrate I and and were used to the proteins. cAMP was in cells treated with H2O2 or isoprenaline using a cAMP to the Myocyte were treated with H2O2 isoprenaline or for 5 min at room Cells were at × g for 5 and the was 100 μl of was added to the and μl of each sample was to a and at room temperature for min. The was with a tissue was homogenized as described but without ml of the was with ml of Triton and centrifuged at × g for 5 min. A was on a ml of using a were reconstituted in SDS sample buffer and for the of the catalytic of PKA by and ml of was prepared as with maleimide buffer and with phosphate-buffered saline plus 1% Triton with protease and overnight at with 100 μl of The affinity was 5 with ml of and reconstituted in nonreducing SDS sample buffer and by protein were as described previously (6Brennan J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, J. P. Wait R. Begum S. Dunn M. J. Eaton P. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) by of using a of of to a ml of were prepared as and with and protease This was for 5 h at with 5 of to α-myosin heavy before at for h with of μl of protein After with the were reconstituted in SDS sample buffer and for the presence of and PKA by tissue from perfused hearts was homogenized as The myofilaments and from a were by × g for 5 and in by and The was into ml of with protease and then of this was with ml of containing mg of type I PKA with or without 100 mm The RI of the kinase was in the oxidized interprotein disulfide and 100 mm to the After at for the myofilaments and were and the was reconstituted in SDS sample buffer. The was then in and centrifuged at × g for 5 min to the myofilaments but the in the of these were with SDS sample and the of the added PKA was using an RI Myocyte were in a on an and using a was in at by a Cells were at and with mm mm 5 mm mm mm mm mm sodium and mm at After a the response to of 100 H2O2 was in the presence and of H89 (1 H89 was used at this as experiments that H89 contractility of myocytes to of PKA of was used for each sample and were cultured and prepared for studies as described previously A. K. R. S. Avkiran M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ARVM were cultured overnight on and to H2O2 (100 μm) or modified Tyrode for 5 min. The myocytes were with and with for min. After cells were with Triton for min. ARVM were again and blocked with 1% for min. After of ARVM were then with 1% containing PKA RI for min. ARVM were then and in the with the to cellular PKA RI. ARVM were then and the were on with medium The were on an with a and were and using between were using of with further using or the multiple were used to amplitude from myocyte contractility was shown are the PKA RI that H2O2 of isolated or cultured ARVM as well as isolated rat hearts the RI of PKA to form an dimer. This dimer formation is by indicating that the is to an disulfide bond (6Brennan J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). formation is with of PKA RI in isolated ARVM after 5 min with 100 were with cultured and in isolated hearts In the we in the of the PKA catalytic PKA a substrate or and are and we phosphorylation of PKA substrates after H2O2 of myocytes. a and a phosphorylation response by and proteins present in ARVM in response to The and substrate proteins were to be and troponin which was using to these proteins that H2O2 (100 μm) phosphorylation of and troponin but is two other PKA substrates proteins are These proteins are to be the PKA substrate and the myofilament protein protein as these with to these proteins that the PKA inhibitor H89 (10 μm) blocked the phosphorylation of PKA substrate proteins. isoprenaline activation of the substrate this was not as a result of the In separate experiments we used isoprenaline and H89 at H89 (1 μm) blocked We a of substrate phosphorylation when PKA was with cAMP after H2O2 of ARVM with isoprenaline a change in to increased cAMP In H2O2 not in in these PKA RI PKA RI subcellular in the isolated after with 100 H2O2 for 1–10 min. PKA RI is a that during H2O2 The RI dimer to the myofilament with evidence on of a in the This RI translocation was in ARVM after H2O2 without translocation of PKA RII not disulfide formation or subcellular This translocation was in studies shown in There is a translocation of PKA RI to the of myocytes treated with This increased was by a in as by an in the of the However, the translocation is and the enhanced is a and is a when of adult ventricular myocytes. We this increased affinity of the RI disulfide dimer for the in in PKA RI in the oxidized but not the has affinity with the This the of disulfide formation for subcellular during H2O2 of PKA the of the kinase using as the during activation. 5 the of the containing the catalytic of PKA in isolated hearts treated with H2O2 and a sample with conditions the as a in H2O2 the with the catalytic in evidence that oxidation in to interprotein disulfide formation the kinase. of the regulatory subunits in these was However, these subunits as of oxidation the only from to during kinase activation and The is to this and as is in the of the regulatory between and investigated the possibility that formation of the PKA RI disulfide dimer to increased affinity with a substrate or associated binding as a that kinase and an for the we used to the regulatory of PKA with proteins that are associated after H2O2 a of the with a PKA RI binding of both the form and oxidized disulfide form of RI. a of affinity of a protein as the H2O2 This protein was as in separate experiments and, as a for this protein as an In experiments we to further its with RI as we an increased of with RI in ARVM in a H2O2 were with from isolated ARVM in to has a well in the response to β-adrenergic which in the increased of contraction. This occurs phosphorylation of the proteins we have shown which were by PKA in response to that H2O2 contractility of cardiac myocytes. the for the amplitude of in the ARVM treated with H2O2 with or without a pretreatment with H2O2 (100 μm) increased the amplitude of μm) with myocytes This of amplitude was to H89 (1 indicating the in are Here we demonstrate that the regulatory of type I PKA has cysteinyl thiols that the cellular redox cellular H2O2 causes the two RI subunits of the to form interprotein This disulfide was but to was a structural bond (10Banky P. Huang L. J. Taylor S. S. J. Biol. Chem. 1998; 273: 35048-35055Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, D. A. Herberg F. W. Banky P. Taylor S. S. J. Biol. Chem. 1997; 272: 28431-28437Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). RI has been as a dimer of its oxidant forming a disulfide bond on to during We previously that PKA RI in the in only forming the disulfide dimer in response to (6Brennan J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). We these as were prepared with which the group and prevents oxidative disulfide The of the RI where the dimer occurs contains the cysteine residues and -38 in rat) that form This N-terminal is at the from the cAMP binding which are in dissociation and activation. Our studies with that both the and oxidized disulfide forms of the RI cAMP. The oxidative disulfide formation in RI is associated with subcellular translocation of type I PKA from the to the and myofilament and to a a as by of subcellular of tissue and of myocytes In this is of that Sci. PubMed Scopus Google Scholar) that a difference between cardiac RI and that associated with the was that the a of disulfide bond formation in the N-terminal they was not the for However, that the presence of the disulfide is important for subcellular This is by phosphorylation of established PKA substrate proteins in these including troponin protein C, and These phosphorylation events are to the inhibitor H89, phosphorylation is mediated by PKA. be by but these have to established PKA substrates and at the be to is notable that the of phosphorylation using the is of H2O2 or isoprenaline the by activation of type II PKA. is that studies the of or both of the cysteines in RI to disulfide formation to kinase activation. However, such is to with that alter the RI dimer constitutively the kinase the RI was this is the AKAP binding cysteine have with these further to the of oxidant-induced activation disulfide possibility was that the H2O2 to cAMP and PKA, as with However, was in cAMP after H2O2 with studies of by oxidants S. Cell. Biochem. 1998; PubMed Scopus (34) Google Scholar, Biochem. Biophys. PubMed Scopus Google Scholar). disulfide formation activates type I PKA without an in cAMP, and this is associated with subcellular to containing PKA of the of PKA after H2O2 we the of the enzyme by Our of these is that conditions the catalytic is part of an two catalytic and two regulatory and, in After H2O2 the catalytic is from the and is to in its in The in PKA the dissociation of the catalytic from the evidence of kinase activation after H2O2 We can these oxidant-induced activation of this kinase in of studies that type I PKA, in to type II, is by substrate-induced sensitization to cAMP (7Vigil D. Blumenthal D. K. Brown S. Taylor S. S. Trewhella J. Biochemistry. 2004; 43: 5629-5636Crossref PubMed Scopus (50) Google Scholar, K. Kopperud R. K. Christensen A. E. Doskeland S. O. J. Biol. Chem. 2005; 280: 13279-13284Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). type I is to at cAMP type the translocation of type I PKA during oxidative stress is important for its activation, the kinase to its physiological We disulfide formation in type I PKA the affinity of the kinase with its substrates or with an associated protein that with AKAPs are a diverse of proteins that of PKA to its multiple and substrates (9Michel J. J. Scott J. D. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 235-257Crossref PubMed Scopus (282) Google Scholar). Type I PKA is and not constitutively associated with AKAP the type II that is to the possibility that the redox of type I PKA its affinity with substrate we the of type I PKA in the or disulfide to cardiac These studies that the but not the form of type I PKA to the myofilament This that disulfide formation in the RI as a redox in both this in binding and in further this we affinity of the regulatory of PKA from or using which we bound and oxidized disulfide forms of RI. We for proteins to from tissue treated with as such proteins an AKAP that the disulfide form of RI to subcellular This was a possibility the regulatory subunits of PKA contain AKAP binding which are in to the redox These binding studies as a protein that in this this protein is not an established AKAP, of with PKA this function. has a well in cardiac but its function as an AKAP is as PKA to its myofilament substrates troponin I and protein C. these proteins were after H2O2 at the as the RI disulfide dimer forms and to the myofilaments Recent studies have that function as AKAPs K. II I J. R. M. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, M. D. R. Scott J. D. J. 2001; PubMed Scopus Google Scholar). The of contains an in a This function found in and to PKA, in this be on RI being in an oxidized disulfide evidence for as an AKAP that localizes oxidized PKA from studies that RI with as the of H2O2 was increased in cells is notable that has been as a protein kinase protein Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google Scholar), further for this as a The in contractility in response to β-adrenergic stimulation is mediated by phosphorylation of substrates by PKA, including proteins we were after H2O2 one of the of H2O2 be increased myocyte which was we This other with H2O2 J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. R. K. K. T. J. Pharmacol. 2003; PubMed Google Scholar), in studies increases in contractility were E. J. Scopus Google Scholar). However, of these studies of contractility were with or for these studies the that oxidants cardiac to the of H2O2 at that the However, in one using to used H2O2 enhanced contractility J. Cell. Full Text PDF PubMed Scopus Google Scholar). we were not to phosphorylation or activation of the in these its activation in cardiac myocytes after H2O2 M. A. M. J. Pharmacol. 1998; Google Scholar). This to increased that further to the of H2O2 The catalytic of PKA is susceptible to cysteine oxidation and is by which is enhanced when the kinase is C. Taylor S. S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, Taylor S. S. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). We have not catalytic S-thiolation but we not in the of the catalytic in of This is in to the of C. Taylor S. S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), found oxidative stress was associated with both interprotein disulfide bond formation to a as well as disulfide which a species and a the We have previously for proteins susceptible to oxidation including S-thiolation (3Eaton P. Byers H. L. Leeds N. Ward M. A. Shattock M. J. J. Biol. Chem. 2002; 277: 9806-9811Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 4Eaton P. Jones M. E. McGregor E. Dunn M. J. Leeds N. Byers H. L. Leung K. Y. Ward M. A. Pratt J. Shattock M. J. J. Am. Soc. Nephrol. 2003; 14: S290-S296Crossref PubMed Google Scholar, A. T. Neubert H. Brennan J. P. Eaton P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17982-17987Crossref PubMed Scopus (243) Google Scholar, J. P. Wait R. Begum S. Bell J. R. Dunn M. J. Eaton P. J. Biol. Chem. 2004; 279: 41352-41360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, J. P. Wait R. Begum S. Dunn M. J. Eaton P. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) and found evidence of catalytic we have to However, is from the studies here that in cardiac cells and to H2O2 activates type I PKA. Our on the of two of PKA. is that type I PKA is primarily to the cellular redox and is by whereas the of type II is the activation in response to cAMP β-adrenergic This type I PKA, which is is to its substrates located in the of PKA independently of increases in cAMP has important for of cellular events can to increases in cellular oxidants such as H2O2 (1Rhee S. G. Bae Y. -S. Lee S. -R. Kwon J. Science's STKE. 2000www. stke. org/cgi/content/full/OCₛigtrans;2000/53/pe1Google Scholar, 2Reth M. Nat. Immunol. 2002; 3: 1129-1134Crossref PubMed Scopus (611) Google Scholar). The oxidant of cells not only increases during of increased but that to activation K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. Lee J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). in this oxidant-induced activation of PKA RI interprotein disulfide formation a mechanism the redox of cells can into are by oxidative and one consequence of is that such have or stimulation of type I PKA, a possibility that further We and Shattock for
Brennan et al. (Tue,) studied this question.