Akt Increases Sarcoplasmic Reticulum Ca2+ Cycling by Direct Phosphorylation of Phospholamban at Thr17
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.036566
ISSN1083-351X
AutoresDaniele Catalucci, Michael V.G. Latronico, Marcello Ceci, Francesca Rusconi, Howard S. Young, Paolo Gallo, Marco Santonastasi, Alfonso Bellacosa, Joan Heller Brown, Gianluigi Condorelli,
Tópico(s)Insect Resistance and Genetics
ResumoCardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca2+ handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca2+ handling. To this end, SR Ca2+ uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr17, the Ca2+-calmodulin-dependent kinase IIδ site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr17. Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca2+ handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy. Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca2+ handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca2+ handling. To this end, SR Ca2+ uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr17, the Ca2+-calmodulin-dependent kinase IIδ site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr17. Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca2+ handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy. Akt, also referred to as protein kinase B, is a serine/threonine kinase found as part of the insulin, insulin-like growth factor-1 (IGF-1) 4The abbreviations used are: IGF-1insulin-like growth factor-1CAMKIIδCa2+-calmodulin-dependent protein kinase IIδCMcardiomyocyteKOknockoutPDK1phosphatidylinositol-dependent kinase-1PKAprotein kinase APLNphospholambanSERCA2asarcoplasmic reticulum Ca-ATPase 2aSRsarcoplasmic reticulumTgtransgenicWTwild typePI3Kphosphatidylinositol 3-kinasePBSphosphate-buffered salineANOVAanalysis of varianceAIPautocamtide 2-related inhibitory peptide. /phosphatidylinositol 3-kinase (PI3K)/phosphatidylinositol-dependent kinase-1 (PDK1) pathway (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar). Upon activation, Akt phosphorylates a broad range of substrates involved in metabolism, transcription, translation, cell growth, differentiation, proliferation, and survival (2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar, 3Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar). In the heart the IGF-1/Akt axis is implicated in the control of physiological cardiac hypertrophy, contractile function, and Ca2+ handling (4McMullen J.R. Shioi T. Zhang L. Tarnavski O. Sherwood M.C. Kang P.M. Izumo S. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12355-12360Crossref PubMed Scopus (454) Google Scholar, 5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar, 6Sun H. 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Cell Biol. 2009; 184: 923-933Crossref PubMed Scopus (94) Google Scholar). insulin-like growth factor-1 Ca2+-calmodulin-dependent protein kinase IIδ cardiomyocyte knockout phosphatidylinositol-dependent kinase-1 protein kinase A phospholamban sarcoplasmic reticulum Ca-ATPase 2a sarcoplasmic reticulum transgenic wild type phosphatidylinositol 3-kinase phosphate-buffered saline analysis of variance autocamtide 2-related inhibitory peptide. The cardiac effects of Akt have been described in many transgenic (Tg) mouse models, and cardiac specific overexpression of different mutated forms of Akt results in varying phenotypes (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar, 12Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (447) Google Scholar, 13Shiojima I. Yefremashvili M. Luo Z. Kureishi Y. Takahashi A. Tao J. Rosenzweig A. Kahn C.R. Abel E.D. Walsh K. J. Biol. Chem. 2002; 277: 37670-37677Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 14Matsui T. Li L. Wu J.C. Cook S.A. Nagoshi T. Picard M.H. Liao R. Rosenzweig A. J. Biol. Chem. 2002; 277: 22896-22901Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 15Kim Y.K. Kim S.J. Yatani A. Huang Y. Castelli G. Vatner D.E. Liu J. Zhang Q. Diaz G. Zieba R. Thaisz J. Drusco A. Croce C. Sadoshima J. Condorelli G. Vatner S.F. J. Biol. Chem. 2003; 278: 47622-47628Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Rota M. Boni A. Urbanek K. Padin-Iruegas M.E. Kajstura T.J. Fiore G. Kubo H. Sonnenblick E.H. Musso E. Houser S.R. Leri A. Sussman M.A. Anversa P. Circ. Res. 2005; 97: 1332-1341Crossref PubMed Scopus (105) Google Scholar). A common effect observed in most of these Tg mouse lines is increased cardiomyocyte (CM) size and either maintained or improved cardiac function (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar, 12Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (447) Google Scholar, 13Shiojima I. Yefremashvili M. Luo Z. Kureishi Y. Takahashi A. Tao J. Rosenzweig A. Kahn C.R. Abel E.D. Walsh K. J. Biol. Chem. 2002; 277: 37670-37677Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 14Matsui T. Li L. Wu J.C. Cook S.A. Nagoshi T. Picard M.H. Liao R. Rosenzweig A. J. Biol. Chem. 2002; 277: 22896-22901Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), with modest or no activation of either mitogen-activated protein kinases (MAPKs) or fetal genes, two hallmarks of maladaptation to stress (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar, 12Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (447) Google Scholar, 13Shiojima I. Yefremashvili M. Luo Z. Kureishi Y. Takahashi A. Tao J. Rosenzweig A. Kahn C.R. Abel E.D. Walsh K. J. Biol. Chem. 2002; 277: 37670-37677Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Our group has previously described a Tg mouse model (Akt-E40K Tg) expressing an Akt with a mutation (E40K) located in its pleckstrin homology domain. This mutation renders Akt constitutively active in a manner similar to stimulation with growth factor (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar). The occurrence of increased CM size with preservation of cardiac contractility constitutes an intriguing aspect of this mouse model. Single cell studies revealed not only increased inotropism but also enhanced lusitropism and an increase in systolic calcium (Ca2+) transients in adult CMs isolated from this Tg mouse (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar, 15Kim Y.K. Kim S.J. Yatani A. Huang Y. Castelli G. Vatner D.E. Liu J. Zhang Q. Diaz G. Zieba R. Thaisz J. Drusco A. Croce C. Sadoshima J. Condorelli G. Vatner S.F. J. Biol. Chem. 2003; 278: 47622-47628Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Enhanced contractility secondary to improved Ca2+ handling is also a feature of a Tg mouse line with nuclear overexpression of Akt (16Rota M. Boni A. Urbanek K. Padin-Iruegas M.E. Kajstura T.J. Fiore G. Kubo H. Sonnenblick E.H. Musso E. Houser S.R. Leri A. Sussman M.A. Anversa P. Circ. Res. 2005; 97: 1332-1341Crossref PubMed Scopus (105) Google Scholar). However, in contrast to the Akt-E40K Tg mouse, an increase in protein kinase A (PKA) activity inducing phosphorylation of phospholamban (PLN) at residue Ser16 was reported to occur in that model. These features raise relevant questions regarding the specific mechanisms by which Akt controls Ca2+ handling. At the sarcoplasmic reticulum (SR) the activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, a pump that transports Ca2+ from the cytosol to the SR lumenal space, is dependent on PLN. In its dephosphorylated state, PLN binds to SERCA2a and inhibits Ca2+ pump activity, whereas phosphorylation of PLN disrupts the interaction with SERCA2a, relieving Ca2+ pump inhibition and enhancing relaxation rate and contractility (17MacLennan D.H. Kranias E.G. Nat. Rev. Mol. Cell Biol. 2003; 4: 566-577Crossref PubMed Scopus (780) Google Scholar). Relief from the inhibitory effects of PLN is the principal contributor to the positive inotropic and lusitropic effects of β-adrenergic agonists (17MacLennan D.H. Kranias E.G. Nat. Rev. Mol. Cell Biol. 2003; 4: 566-577Crossref PubMed Scopus (780) Google Scholar). The activity of PLN is inhibited by protein kinase A (PKA) and Ca2+-calmodulin-dependent protein kinase (CAMK)IIδ, which phosphorylate PLN at Ser16 and Thr17, respectively (17MacLennan D.H. Kranias E.G. Nat. Rev. Mol. Cell Biol. 2003; 4: 566-577Crossref PubMed Scopus (780) Google Scholar). Phosphorylation of PLN by PKA was demonstrated to be a prerequisite for subsequent phosphorylation by CAMKIIδ following β-adrenergic agonist stimulation (18Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Phosphorylation of PLN at Ser16 was also reported to be sufficient to mediate the inotropic responses of the heart to β-adrenergic agonists (19Chu G. Lester J.W. Young K.B. Luo W. Zhai J. Kranias E.G. J. Biol. Chem. 2000; 275: 38938-38943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, during other types of stimuli, such as ischemia or frequency-dependent stimuli, Thr17, but not Ser16, becomes phosphorylated, suggesting that phosphorylation at Thr17 is more relevant than that of Ser16 under certain conditions (17MacLennan D.H. Kranias E.G. Nat. Rev. Mol. Cell Biol. 2003; 4: 566-577Crossref PubMed Scopus (780) Google Scholar, 20Hagemann D. Kuschel M. Kuramochi T. Zhu W. Cheng H. Xiao R.P. J. Biol. Chem. 2000; 275: 22532-22536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Moreover, it was recently reported that PLN phosphorylation at Ser16 is determined by increased protein levels of adenyl cyclase VI (21Gao M.H. Tang T. Guo T. Miyanohara A. Yajima T. Pestonjamasp K. Feramisco J.R. Hammond H.K. J. Biol. Chem. 2008; 283: 33527-33535Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). This adenyl cyclase overexpression was shown to be associated not with PKA activation but rather with phosphorylation of Akt and its substantial relocalization to the nucleus that, similar to results obtained in nuclear Akt Tg mice (16Rota M. Boni A. Urbanek K. Padin-Iruegas M.E. Kajstura T.J. Fiore G. Kubo H. Sonnenblick E.H. Musso E. Houser S.R. Leri A. Sussman M.A. Anversa P. Circ. Res. 2005; 97: 1332-1341Crossref PubMed Scopus (105) Google Scholar), resulted in Ser16 PLN phosphorylation. In this study, we demonstrate that upon physiological activation Akt translocates to the SR where it interacts with and phosphorylates PLN at Thr17. This leads to improved contractility without stimulating hypertrophic growth normally associated with Akt. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. The generation of Tg mice with cardiac specific overexpression of constitutively active Akt (Akt-E40K) has been described previously (5Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12333-12338Crossref PubMed Scopus (402) Google Scholar). Cardiac specific PDK1-inducible knock-out (KO) mice (MerCreMer α-MHC PDK1) were generated as described elsewhere (11Catalucci D. Zhang D.H. DeSantiago J. Aimond F. Barbara G. Chemin J. Bonci D. Picht E. Rusconi F. Dalton N.D. Peterson K.L. Richard S. Bers D.M. Brown J.H. Condorelli G. J. Cell Biol. 2009; 184: 923-933Crossref PubMed Scopus (94) Google Scholar). Adult and neonatal mouse CMs were isolated according to published procedures (15Kim Y.K. Kim S.J. Yatani A. Huang Y. Castelli G. Vatner D.E. Liu J. Zhang Q. Diaz G. Zieba R. Thaisz J. Drusco A. Croce C. Sadoshima J. Condorelli G. Vatner S.F. J. Biol. Chem. 2003; 278: 47622-47628Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 22Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Adult CMs were from age-matched mice between 8 and 13 weeks of age. 1-Day-old neonatal mouse CMs were loaded with the Ca2+ indicator fura 4-AM (Molecular Probes, Eugene, OR) by incubating the cells in Tyrode's solution (in mm, 121 NaCl, 4.6 KCl, 1.2 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 15 glucose, and 10 HEPES, pH 7.4) containing 2 μmol/liter fura 4-AM for 20 min. The cells were then washed with Tyrode's solution and incubated for 30 min. One more wash was performed before mounting the chamber on an epifluorescence inverted microscope (Nikon TS100) with an attached CCD camera (Ionoptix MyoCam, Ionoptix, Milton, MA). The coverslip was superfused with Tyrode's solution at room temperature at a rate of 20 ml/h. Cells were stimulated using an external stimulator, and Ca2+ concentration was measured using a dual excitation spectrofluorometer that detects the fluorescence excited by UV light at 360 and 380 nm with an emission at 505 nm. Homogenization of pulverized ventricular tissue from WT and Tg mouse hearts, as well as isolation of SR-enriched microsomes and cytosolic fractions, was done according to published procedures (23Said M. Vittone L. Mundina-Weilenmann C. Ferrero P. Kranias E.G. Mattiazzi A. Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1198-H1205Crossref PubMed Scopus (48) Google Scholar, 24Netticadan T. Temsah R. Osada M. Dhalla N.S. Am. J. Physiol. 1999; 277: C384-C391Crossref PubMed Google Scholar). SR Ca2+ uptake assays were performed on ventricular homogenates at room temperature based on a protocol by Pagani and Solaro (25Pagani E.D. Solaro R.J. Methods Pharmacol. 1984; 5: 49-61Google Scholar) and modified in the Dillmann laboratory (26Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). When used, 40 μm Akt-IV (Calbiochem), 5 μm AIP, or 5 μm KN93 was preincubated with homogenized aliquots at room temperature before adding ATP. CMs were cultured in serum-free medium for 3 h prior to stimulation with 0.1 mm insulin for 40 min. When necessary, cells were pretreated with inhibitors (0.1 mm KN93 or 10 μm AKT inhibitor IV) for 10 min. These reagents were obtained from Calbiochem. At the end of the stimulation period, cells were rinsed in ice-cold PBS and scraped on ice in lysis buffer (in mm, 150 NaCl, 50 Tris, pH 7.5, 30 NaF, 0.1 Na3VO4, and 1 phenylmethylsulfonyl fluoride) containing 1% Triton X-100 and a protease inhibitor mixture (Complete, Roche Applied Science). Lysates were cleared (14,000 rpm for 10 min at 4 °C) and analyzed by Western blotting. Hearts from Tg and WT mice were homogenized at 4 °C in 1 ml of homogenization buffer (in mm, 25 imidazole, pH 7.0, 150 KCl, containing 0.1% Triton, 0.1% Nonidet P-40, and protease inhibitor mixture) with a Teflon glass Thomas tissue grinder. After a 20-min centrifugation at 14,000 rpm at 4 °C, 50 ml of mono-HA11 beads (Covance Babco, affinity matrix mono-HA11) was added to the supernatant and incubated for 60 min at 4 °C with gentle shaking. For coimmunoprecipitation experiments, beads were washed three times with homogenization buffer and then resuspended in 5× sample buffer without reducent. Samples were boiled and loaded onto SDS-PAGE. For in vitro kinase assay and SR uptake, beads were washed with homogenization buffer and added to the homogenized heart obtained from WT mice in kinase buffer (in mm, 25 imidazole, 10 MgCl2, 0.1 sodium vanadate, 20 NaF, and 0.05 ATP). Reaction mixtures were incubated for 25 min at 30 °C. Subsequently, beads were resuspended in 5× sample buffer without reducent for Western blot analysis or used to measure SR Ca2+ uptake, as described above. An in vitro Akt kinase assay was performed as follows: recombinant PLN was obtained as described previously (27Douglas J.L. Trieber C.A. Afara M. Young H.S. Protein Expr. Purif. 2005; 40: 118-125Crossref PubMed Scopus (37) Google Scholar), whereas purified active Akt was purchased from Cell Signaling (Waltham, MA). The kinase assay was performed according to the manufacturer's instructions, with or without active Akt; detection of total PLN and PLN phosphorylated at Ser16 or Thr17 was performed by Western blot analysis. PKA activity was measured using a cAMP assay kit (Upstate, Charlottesville, VA) according to the manufacturer's instructions. SDS-PAGE and Western blot analysis were performed on total cytosolic lysate and the SR fraction from WT, Tg, or KO mouse ventricles. The following antibodies were used: anti-total PLN and anti-phospho Thr17 PLN antibodies (Badrilla, Leeds, UK); anti-phospho-Ser16 PLN antibody (Upstate, Charlottesville, VA); anti-phospho-Ser473 Akt and anti-total Akt antibodies (Cell Signaling); anti-phospho-Thr287 CAMKIIδ antibody (Affinity Bioreagents, Golden, CO); anti-total CAMKIIδ antibody (kindly provided by Dr. Harold Singer) (28Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer H.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar); and anti-PDK1 antibody (Calbiochem). Three adenoviruses were produced. One (mock) was an infective virus with no transgene expression. The second (Ad Akt-E40K) contained the HA-tagged E40K Akt transgene used for production of our Tg mouse line. The third (Ad Akt-PLN) contained an active form of Akt linked to an SR localization sequence at the C terminus. Constitutive activity of the T308D/S473D Akt mutant, in which the aspartic acid residues mimic phosphorylation, has been described previously (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar, 29Bellacosa A. Chan T.O. Ahmed N.N. Datta K. Malstrom S. Stokoe D. McCormick F. Feng J. Tsichlis P. Oncogene. 1998; 17: 313-325Crossref PubMed Scopus (456) Google Scholar). This mutant is biologically active and able to mediate some of the effects of Akt, including transformation of chicken embryo fibroblasts (30Aoki M. Batista O. Bellacosa A. Tsichlis P. Vogt P.K. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 14950-14955Crossref PubMed Scopus (258) Google Scholar). To serve as an SR localization signal, amino acids 23–52 of the transmembrane region of PLN were used. In addition, this PLN sequence contained mutations L31A and N34A to eliminate its ability to inhibit SERCA activity. This sequence was obtained from the αMHC-AIP4-SRL vector (kindly provided by Dr. J. R. Dedman, University of Cincinnati College of Medicine, Cincinnati, OH) (31Ji Y. Li B. Reed T.D. Lorenz J.N. Kaetzel M.A. Dedman J.R. J. Biol. Chem. 2003; 278: 25063-25071Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, the Ad Akt-PLN vector was obtained by homologous recombination following cotransformation of a 3′Akt-PLN fragment and Ad Akt-E40K vector into Escherichia coli Bj5183. Three PCR steps were used to amplify the 3′Akt-PLN fragment. First, 5′L-Akt-PLN (5′-GCGGATTCCCCGTGCCAAGAGTGAGATCTTCCGTTTATGTACCCATACGATGTTCCAGATTACG-3′) and 3′L-Akt-PLN (5′-GATAAATAGGTTCTGGAGATTCTGACGTGCTTGGGCGAGGGCTGTGCCACTGGCTGAGTAGGAG-3′) as well as PLN_5_fw (5′-CACAGCCCTCGCCCAAGCACGTCAGAATCTCCAGAAC-3) and 3′l-PLN-NLS (5′-GATCCGTCGACTCTAGATTTAAATGTTTCGTCTAGATCACAGAAGCATCACAATGATGCAGATCAGCAG-3′) primers were used to amplify Akt and PLN sequences, respectively. Subsequently, because of an introduced overlapping sequence of obtained PCR products, the 3′Akt-PLN fusion fragment was obtained with a final PCR amplification. Finally, viral vectors were amplified and purified in 3% sucrose/PBS by ViraQuest, Inc. (North Liberty, IA). Isolated Ca2+-tolerant CMs were plated on laminin-coated slides and treated as follows: (i) infected with mock (empty vector) or recombinant adenoviral vectors at 100 multiplicity of infection for 1 h and then cultured in serum-free medium for an additional 48 h; (ii) incubated with insulin for 30 min. After the aforementioned treatments, cells were fixed in 4% paraformaldehyde, rinsed in PBS, and permeabilized with blocking buffer for 20 min (0.1% Triton X-100, 1% normal donkey serum, 1% cold-water fish gelatin, and 20 mm glycine). Cells were then costained overnight at 4 °C with anti-PLN antibody (1:200) and either anti-Akt antibody (1:50) or anti-HA tag antibody (1:50) in diluted working buffer (blocking buffer diluted 10× with PBS). After extensive washing, cells were incubated with fluorescently labeled secondary antibodies (anti-mouse rhodamine from Molecular Probes and anti-rabbit fluorescein isothiocyanate from Sigma) for 1 h at 4 °C in the dark. Subsequently, cells were washed extensively with working buffer and mounted. Slides were stored at 4 °C until imaged using a Bio-Rad Radiance 2000 Confocal/2-Photon microscope with a ×60 Plan-apochromat 1.4 n.a. objective (Zeiss). Individual images (1024 × 1024) were converted to a tiff format and merged as pseudocolor RGB images using Imaris (Bitplane AG). Experiments were repeated at least twice to confirm the fluorescence patterns. 1-Day-old neonatal mouse CMs were plated in serum-free medium. 24 h after plating CMs were infected with mock, Ad Akt-E40K, or Ad Akt-PLN at a multiplicity of infection of 50. 48 h later, 2.5 μCi/ml [3H]leucine was added to the medium. The assay was terminated after 12 h by washing the cells three times with ice-cold phosphate-buffered saline without Ca2+ and Mg2+ and fixing in ice-cold 5% trichloroacetic acid. Thereafter, proteins were solubilized in 0.5 n NaOH, and radioactivity and protein content were measured. Group data are given as mean ± S.D. and compared using Student's t test or one-way ANOVA as indicated. Analysis was performed using GraphPad Prism version 4.0 for Mac (GraphPad Software, San Diego). We decided first to determine SERCA2a activity in Tg mice by analyzing the phosphorylation status of PLN at Ser16 and Thr17 by Western blotting. We found that in Tg heart extracts phosphorylation of PLN at Thr17 was increased when compared with WT, whereas phosphorylation at Ser16 was unchanged or even reduced (Fig. 1A). The partial reduction of PLN phosphorylation at Ser16 in Tg could be explained by a decrease in PKA activity that we found in these mice compared with WT, as evaluated by measurement of the level of cAMP (0.270 ± 0.025 versus 0.470 ± 0.015 pmol/mg protein, p < 0.05, respectively). Because phosphorylation at Thr17 is an indirect measurement of CAMKIIδ activity, we hypothesized that CAMKIIδ activity might be higher in the hearts of Tg than WT. To test this, cytosolic and SR extracts were probed with antibodies for total and active, Thr286-phosphorylated CAMKIIδ. Surprisingly, phosphorylation of CAMKIIδ at Thr286 was decreased in Tg compared with WT (Fig. 1B). This indicated that CAMKIIδ activity is reduced in Tg mice hearts and, consequently, plays a minor, if any, role in phosphorylation of PLN at Thr17 in this model. The consensus sequence of PLN for CAMKIIδ (RXXT) is similar to the canonical consensus site sequence for Akt phosphorylation (RXRXX(S/T)). We therefore speculated that the Thr17 residue of PLN could also be phosphorylated by Akt. To test this, we first analyzed KO mice harboring a cardiac specific deletion of PDK1, the upstream activator of Akt (11Catalucci D. Zhang D.H. DeSantiago J. Aimond F. Barbara G. Chemin J. Bonci D. Picht E. Rusconi F. Dalton N.D. Peterson K.L. Richard S. Bers D.M. Brown J.H. Condorelli G. J. Cell Biol. 2009; 184: 923-933Crossref PubMed Scopus (94) Google Scholar). In this model, all three isoforms of Akt are dephosphorylated. We found that phosphorylation at Thr17 was reduced in KO compared with WT, whereas there was no apparent change in phosphorylation at Ser16 (Fig. 2). Revealingly, CAMKIIδ activity, as assessed by the level of phosphorylation at Thr286, was comparable in KO and WT (Fig. 2). These results corroborated the hypothesis that Akt might be involved in PLN phosphorylation at Thr17. Direct phosphorylation of PLN by Akt implies that these two proteins colocalize and physically interact with each other at the SR. In fact, Western blotting with both anti-HA-tagged and anti-phospho-Akt antibodies revealed that Akt-E40K was indeed found at the SR in Tg (Fig. 3A). Importantly, Akt translocates to the SR also in a more physiological setting, i.e. treatment of WT CMs with insulin. In fact, confocal microscopy images of in vitro WT CMs stained for total Akt revealed a rather homogeneous and diffuse staining in untreated CMs but a striated pattern, which was superimposed to that for PLN, after insulin stimulation (Fig. 3B). Further evidence for direct interaction between Akt and PLN came from pulldown experiments in which Akt-E40K was immunoprecipitated from heart extracts with an anti-HA antibody and PLN probed for Western blotting with an anti-PLN antibody. We found that in Tg extracts, PLN coprecipitated with Akt-E40K (Fig. 3C). In addition, in an in vitro kinase assay in which recombinant full-length PLN was incubated with or without a commercially available active Akt, PLN became phosphorylated at Thr17 but not at Ser16 in the presence of active Akt (Fig. 3D). Moreover, stimulation of CMs with insulin in the presence of specific Akt or CAMKII inhibitors suggested that CAMKIIδ and Akt concur in phosphorylating PLN at Thr17 (Fig. 4). The effect of Akt on SR Ca2+ handling was assessed by an SR 45Ca2+ uptake ass
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