An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Abstract

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein β (mAKAPβ) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPβ selectively binds type 5 AC in the heart and that mAKAPβ-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPβ-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245–340), and expression of this peptide disrupts endogenous mAKAPβ-AC association. Accordingly, disruption of mAKAPβ-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPβ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy. Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein β (mAKAPβ) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPβ selectively binds type 5 AC in the heart and that mAKAPβ-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPβ-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245–340), and expression of this peptide disrupts endogenous mAKAPβ-AC association. Accordingly, disruption of mAKAPβ-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPβ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy. The formation of multimolecular protein complexes contributes to the specificity of intracellular signaling pathways, including those regulating cardiac myocyte hypertrophy. The cAMP-dependent protein kinase (PKA) 3The abbreviations used are: PKAcAMP-dependent protein kinaseAKAPprotein kinase A-anchoring proteinGSTglutathione S-transferaseACadenylyl cyclaseNEnorepinephrineEIAenzyme immunoassayGTPγSguanosine 5′-3-O-(thio)triphosphateβ-galβ-galactosidase. is targeted to specific intracellular domains by protein kinase A-anchoring proteins (AKAPs) that often serve as scaffolding proteins for diverse signaling enzymes (1.McConnachie G. Langeberg L.K. Scott J.D. Trends Mol. Med. 2006; 12: 317-323Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In the heart, global disruption of PKA anchoring affects cardiac contractility, while the inhibited expression of individual AKAPs such as mAKAPβ or AKAP-Lbc attenuates adrenergic-induced hypertrophy of cultured neonatal myocytes (2.Fink M.A. Zakhary D.R. Mackey J.A. Desnoyer R.W. Apperson-Hansen C. Damron D.S. Bond M. Circ. Res. 2001; 88: 291-297Crossref PubMed Scopus (119) Google Scholar, 3.Appert-Collin A. Cotecchia S. Nenniger-Tosato M. Pedrazzini T. Diviani D. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10140-10145Crossref PubMed Scopus (84) Google Scholar, 4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar). We have recently shown that specific AKAPs, namely AKAP79 and Yotiao, bind adenylyl cyclases (AC) (5.Bauman A.L. Soughayer J. Nguyen B.T. Willoughby D. Carnegie G.K. Wong W. Hoshi N. Langeberg L.K. Cooper D.M. Dessauer C.W. Scott J.D. Mol. Cell. 2006; 23: 925-931Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google Scholar). However, the functional significance of AC-AKAP complexes has not been demonstrated. cAMP-dependent protein kinase protein kinase A-anchoring protein glutathione S-transferase adenylyl cyclase norepinephrine enzyme immunoassay guanosine 5′-3-O-(thio)triphosphate β-galactosidase. mAKAPβ, expressed in striated myocytes, is one of two known splice variants encoded by the single mAKAP (AKAP6) gene (7.Michel J.J. Townley I.K. Dodge-Kafka K.L. Zhang F. Kapiloff M.S. Scott J.D. Mol. Cell. 2005; 20: 661-672Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We previously published that mAKAPβ is primarily localized to the outer membrane of the nuclear envelope via direct binding to nesprin-1α (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar, 8.Kapiloff M.S. Schillace R.V. Westphal A.M. Scott J.D. J. Cell Sci. 1999; 112: 2725-2736Crossref PubMed Google Scholar). In cardiac myocytes, mAKAPβ serves as the scaffold for a multimolecular signaling complex that in addition to PKA includes the ryanodine receptor (RyR2), the protein phosphatases PP2A and calcineurin, phosphodiesterase 4D3 (PDE4D3), exchange protein activated by cAMP (Epac1), ERK5, and MEK5 mitogen-activated protein kinases, molecules implicated in the regulation of cardiac hypertrophy (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar, 7.Michel J.J. Townley I.K. Dodge-Kafka K.L. Zhang F. Kapiloff M.S. Scott J.D. Mol. Cell. 2005; 20: 661-672Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8.Kapiloff M.S. Schillace R.V. Westphal A.M. Scott J.D. J. Cell Sci. 1999; 112: 2725-2736Crossref PubMed Google Scholar, 9.Carlisle Michel J.J. Dodge K.L. Wong W. Mayer N.C. Langeberg L.K. Scott J.D. Biochem. J. 2004; 381: 587-592Crossref PubMed Scopus (95) Google Scholar, 10.Dodge K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (403) Google Scholar, 11.Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (442) Google Scholar, 12.Kapiloff M.S. Jackson N. Airhart N. J. Cell Sci. 2001; 114: 3167-3176Crossref PubMed Google Scholar, 13.Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1712) Google Scholar). mAKAPβ complexes facilitate cross-talk between MAP kinase, calcium, and cAMP signaling pathways, permitting feedback inhibition of cAMP levels and the dynamic regulation of PKA and ERK5 activity (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar, 9.Carlisle Michel J.J. Dodge K.L. Wong W. Mayer N.C. Langeberg L.K. Scott J.D. Biochem. J. 2004; 381: 587-592Crossref PubMed Scopus (95) Google Scholar, 10.Dodge K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (403) Google Scholar, 11.Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (442) Google Scholar, 12.Kapiloff M.S. Jackson N. Airhart N. J. Cell Sci. 2001; 114: 3167-3176Crossref PubMed Google Scholar, 13.Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1712) Google Scholar). Accordingly, mAKAPβ RNAi attenuates adrenergic and cytokine-induced hypertrophy of cultured rat neonatal ventricular myocytes (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar, 11.Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (442) Google Scholar). Because mAKAPβ forms a complex with two cAMP effectors and a metabolizing enzyme for cAMP, we considered whether AC might also be an integral part of the mAKAPβ complex. We now demonstrate that type 5 adenylyl cyclase (AC5) binds directly a unique N-terminal site on mAKAPβ and is the predominant AC isoform associated with mAKAPβ in the heart. We show that AC5 bound to mAKAPβ is inhibited by PKA-dependent negative feedback. Importantly, inhibition of endogenous mAKAPβ-AC5 binding revealed the functional importance of these complexes for the regulation of cAMP-dependent myocyte hypertrophy. Antibodies were obtained as described under supplemental methods. The mAKAPβ isoform is identical to residues 245–2314 of mAKAPα. Thus, all mutants are numbered according to mAKAPα. The mammalian expression plasmids for wild type and ΔPKA (deletion of residues 2053–2073) mAKAPβ are as previously described (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar). Adenoviruses that express C-terminally Myc-tagged mAKAP 1–587, 245–340 (Myc-AC-BD), 245–587, 1286–1833, and 1835–2312 fragments were generated using the pTRE shuttle vector and the Adeno-X Tet-off System (Clontech) as previously described for Myc-mAKAP-(585–1286) (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar). Encoded proteins are expressed in a doxycycline-inhibited manner when co-infected with adenovirus that constitutively express the tetracycline-controlled transactivator (tTA). All other expression vectors for AC isoforms and/or AKAP79 are described in supplemental methods. Frozen adult rat hearts (Pel-Freez Biologicals) or AC5+/+ and AC5−/− mouse hearts (generously provided by Kirk Hammond) were thawed in phosphate-buffered saline with protease inhibitors. The hearts were rinsed, diced, and homogenized with a polytron in lysis buffer lacking detergent. After addition of 1.0% Triton X-100, the extracts were further homogenized with a Dounce homogenizer, followed by centrifugation at 14,000 rpm to remove the insoluble fraction. For competition experiments, GST fusion proteins were added to the final homogenization step. The extracts were used immediately for experiments. For immunoprecipitations, extracts were incubated with 10 μl of serum and 20 μl of protein G-agarose for 3 h at 4 °C. The beads were washed twice with buffer containing 0.05% lubrol before measurement of AC activity as described in detail in the on-line supplement (6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google Scholar, 14.Dessauer C.W. Chen-Goodspeed M. Chen J. J. Biol. Chem. 2002; 277: 28823-28829Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cardiac myocytes or transfected COS-7 cells were rinsed with phosphate-buffered saline and then lysed with lysis buffer (50 mm HEPES pH 7.4, 1 mm EDTA, 1 mm MgCl2, 150 mm NaCl, 0.5% C12E9, protease inhibitors). Cellular debris was removed by centrifugation, and an aliquot was removed to measure AC activity in the starting lysates. The clarified extracts were then subjected to immunoprecipitation followed by an AC assay upon addition of 50 nm Gαs-GTPγS and 100 μm forskolin, as detailed in the on-line supplement and previously described (6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google Scholar, 14.Dessauer C.W. Chen-Goodspeed M. Chen J. J. Biol. Chem. 2002; 277: 28823-28829Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). GST fusion proteins (50 μg) were incubated with heart extract (∼1/3 heart per sample) for 30 min. Glutathione-agarose (30 μl of packed resin) was added for an additional 2 h. Samples were washed three times with wash buffer and assayed for AC activity directly on the resin as described in the on-line supplement (14.Dessauer C.W. Chen-Goodspeed M. Chen J. J. Biol. Chem. 2002; 277: 28823-28829Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Ventricular myocytes (over 90% free of fibroblasts) were prepared from 2–3-day-old Sprague-Dawley rats, as previously described (8.Kapiloff M.S. Schillace R.V. Westphal A.M. Scott J.D. J. Cell Sci. 1999; 112: 2725-2736Crossref PubMed Google Scholar). Adenoviral infection, immunocytochemistry, and [3H]leucine incorporation were performed as described in the on-line supplement. All data are expressed as means ± S.E. of the mean. Each n represents the results of individual experiments using separate primary cultures. p values were calculated using a two-tailed, paired Student's t test. To test whether AC associates with mAKAPβ, protein complexes were immunoprecipitated from adult rat heart extracts using multiple mAKAP antisera and were assayed for AC activity (IP-AC assay) (Fig. 1A). AC activity associated with each mAKAP sera was 3-fold greater than the respective preimmune control sera, representing 1.5 ± 0.8% of the total AC activity in heart extracts. IP-AC assays using rat neonatal ventricular myocytes confirmed the association of AC and mAKAPβ in the heart (Fig. 1B). mAKAP antiserum precipitated 10 ± 3% of the total AC activity in cardiac myocytes. In cardiac myocytes, nesprin-1α anchors mAKAPβ at the nuclear envelope (15.Pare G.C. Easlick J.L. Mislow J.M. McNally E.M. Kapiloff M.S. Exp. Cell Res. 2005; 303: 388-399Crossref PubMed Scopus (93) Google Scholar). AC was also immunoprecipitated from adult rat heart extract with a nesprin-1 antiserum (Fig. 1A). Detection of mAKAPβ and nesprin-1α in the mAKAP and nesprin-1 immunoprecipitates served as controls (supplemental Fig. S1). To determine whether AC association with nesprin-1α was direct or indirect through mAKAPβ, we expressed mAKAP-(585–1286) in cultured neonatal myocytes (Fig. 2A) to competitively inhibit endogenous mAKAPβ-nesprin-1α binding (15.Pare G.C. Easlick J.L. Mislow J.M. McNally E.M. Kapiloff M.S. Exp. Cell Res. 2005; 303: 388-399Crossref PubMed Scopus (93) Google Scholar). AC activity associated with nesprin-1 co-immunoprecipitation was blocked by expression of mAKAP-(586–1286), but not by a β-galactosidase (β-gal) control (Fig. 1C). Thus, AC associates with mAKAPβ bound to nesprin-1α at the cardiac myocyte nuclear envelope. AC5 and AC6 are the most abundant AC isoforms in cardiac myocytes (16.Katsushika S. Chen L. Kawabe J. Nilakantan R. Halnon N.J. Homcy C.J. Ishikawa Y. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8774-8778Crossref PubMed Scopus (195) Google Scholar, 17.Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar). Because of the lack of suitable, isoform-specific AC antibodies, we determined which AC isoforms bound mAKAPβ by co-expressing ACs and Myc-tagged mAKAPβ in COS-7 cells. AC5 and AC2 were significantly enriched in α-Myc immunoprecipitates (8-fold and Fig. In AC6 and were not significantly associated with (Fig. and data not The total AC activity in COS-7 extracts confirmed that each AC isoform was expressed (Fig. IP-AC assay of mAKAPβ immunoprecipitates from wild type and AC5 knock-out mouse hearts that AC5 is the AC isoform in (Fig. We the mAKAPβ site that binds endogenous AC by Myc-tagged mAKAP fragments in neonatal myocytes and an IP-AC assay (Fig. AC activity was associated with mAKAPβ and (Fig. was by assays using rat heart extracts and fusion proteins (Fig. and AC activity was precipitated with mAKAP fragments that residues The fusion protein also was to AC activity to a (Fig. To that was for binding GST fusion proteins were used as in IP-AC assays using heart extracts. and inhibited endogenous GST (Fig. AC5 three domains that with mAKAPβ, the N-terminal and the and domains (Fig. R. Dessauer C.W. PubMed Scopus Google Scholar). GST of the three AC5 domains were used to mAKAPβ from lysates. each AC mAKAPβ not we binding to mAKAP with a complex of all three AC5 The of and domains increased mAKAPβ by (Fig. The to be as and and domains in the of (Fig. but not Yotiao, also bind AC5 (5.Bauman A.L. Soughayer J. Nguyen B.T. Willoughby D. Carnegie G.K. Wong W. Hoshi N. Langeberg L.K. Cooper D.M. Dessauer C.W. Scott J.D. Mol. Cell. 2006; 23: 925-931Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google Scholar). To determine whether mAKAPβ and AKAP79 on we whether competitively inhibit Although with endogenous mAKAPβ binding to AC in heart extracts (Fig. and binds AC5 directly (Fig. was to inhibit (supplemental Fig. To of mAKAPβ association on AC5 from COS-7 cells AC5 and/or mAKAPβ were assayed for AC AC5 was inhibited by or upon with or forskolin, (Fig. The inhibition of AC5 by mAKAPβ not to be a direct of protein binding addition of or to on AC5 activity and expression of on AC activity in the absence of mAKAP not of AC5 by mAKAPβ was not to AC5 protein levels (Fig. the inhibition of AC5 by mAKAPβ on PKA association with the complex. of a mAKAPβ to bind PKA ΔPKA (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Fig. 2A) on AC5 activity in (Fig. with published that AC5 is a PKA and to feedback inhibition G. Kawabe J. T. Homcy C.J. Ishikawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. A. J. R. G. R. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). AC2 is not inhibited by PKA and was not inhibited by with mAKAPβ not of AC5−/− revealed that AC5 contributes to the of cardiac in and with L. A. Chen W. S. Ishikawa Y. J. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. Y. S. C. Kawabe J. Ishikawa Y. 2007; PubMed Scopus Google Scholar, S. G. Kawabe J. G. C. J. J. Ishikawa Y. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). that mAKAPβ inhibited AC5 activity (Fig. we that disruption of mAKAPβ-AC5 complexes in cardiac myocytes AC5 activity and myocyte hypertrophy. we expressed in cultured rat neonatal ventricular myocytes using a adenovirus (Fig. of competitively inhibited endogenous mAKAPβ-AC binding in myocytes as by IP-AC assay (Fig. to the competition for in heart extracts (Fig. not AC from endogenous mAKAPβ, but also increased total cAMP levels in myocytes (Fig. myocytes greater cAMP than myocytes ± ± and greater cAMP upon with the adrenergic agonist norepinephrine 10 ± ± Thus, mAKAPβ serves to cAMP levels in myocytes. cAMP signaling is known to hypertrophy. Because disruption of the complex to increased cAMP, we whether increased AC5 activity myocyte were with adenovirus (Fig. a and or and for 2 in the absence a and or and of Consistent with the in cAMP levels (Fig. cells were ± in than cells (Fig. the cells greater a of myocyte hypertrophy (Fig. a separate addition of which protein expression in this between and not results were obtained by expression of in transfected myocytes not total protein was as an additional for cardiac hypertrophy. [3H]leucine incorporation protein was ± greater in myocytes with control (Fig. Although expression increased total cAMP levels in myocytes, expression of the peptide not in further myocyte with that of and results in myocyte in Circ. Res. 2000; PubMed Scopus Google Scholar). In this we show that AC binds directly to mAKAPβ in cardiac myocytes, mAKAPβ as the to bind directly all of the required for cAMP signaling, including enzymes for cAMP (AC), signaling and binds mAKAPβ K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (442) Google AC binding not to with the other of mAKAP complexes for which binding have been as AC nesprin-1α (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (403) Google PKA (8.Kapiloff M.S. Schillace R.V. Westphal A.M. Scott J.D. J. Cell Sci. 1999; 112: 2725-2736Crossref PubMed Google and kinase 1 in (7.Michel J.J. Townley I.K. Dodge-Kafka K.L. Zhang F. Kapiloff M.S. Scott J.D. Mol. Cell. 2005; 20: 661-672Abstract Full Text Full Text PDF PubMed Scopus (59) Google bind mAKAP AC 2 and 5 bind mAKAPβ, the predominant complex in the heart is mAKAPβ-AC5 (Fig. AC2 binding to mAKAP be in or muscle or mAKAPβ are A of specificity was for Yotiao, such that ACs and but not ACs and bound this (5.Bauman A.L. Soughayer J. Nguyen B.T. Willoughby D. Carnegie G.K. Wong W. Hoshi N. Langeberg L.K. Cooper D.M. Dessauer C.W. Scott J.D. Mol. Cell. 2006; 23: 925-931Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google Scholar). The of AC2 has previously been as the site of with (6.Piggott L.A. Bauman A.L. Scott J.D. Dessauer C.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13835-13840Crossref PubMed Scopus (87) Google the of AC5 contributes to its increased binding to mAKAPβ (Fig. Thus, with AC be a for of complexes. we show that multiple domains of AC5 and bind mAKAPβ, as for other AC including and R. Dessauer C.W. PubMed Scopus Google Scholar). mAKAPβ and nesprin-1α at the cardiac myocyte nuclear and AC activity was associated with mAKAPβ and with nesprin-1α (Fig. (15.Pare G.C. Easlick J.L. Mislow J.M. McNally E.M. Kapiloff M.S. Exp. Cell Res. 2005; 303: 388-399Crossref PubMed Scopus (93) Google Scholar). In the cardiac the membrane has that to the and to the outer nuclear membrane with which the is AC5 has been on the and of T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have also been of AC5 at the nuclear envelope C. G. A. N. Res. 2006; PubMed Scopus Google Scholar, S. D.M. G. A. C.J. J. Exp. Google Scholar). that cAMP at cardiac myocytes including associated with nuclear and S. Res. PubMed Scopus Google Scholar). are also targeted to the including and in G. J. Res. 2008; PubMed Scopus Google Scholar). Thus, the mAKAPβ scaffold a localized cAMP signaling complex at the nuclear envelope. but not has been associated with of the AC5 gene the to S. Kawabe J. A. G. C. J. J. Ishikawa Y. Circ. Res. PubMed Scopus Google and the of heart S. G. Kawabe J. G. C. J. J. Ishikawa Y. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). are including cardiac and L. A. Chen W. S. Ishikawa Y. J. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of AC5 to mAKAPβ is with that mAKAPβ complexes cAMP signaling in cardiac myocytes. using mAKAPβ RNAi a for mAKAPβ expression in neonatal myocyte hypertrophy in (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google and the signaling enzymes and ERK5 are associated with mAKAPβ complexes (4.Pare G.C. Bauman A.L. McHenry M. Michel J.J. Dodge-Kafka K.L. Kapiloff M.S. J. Cell Sci. 2005; 118: 5637-5646Crossref PubMed Scopus (106) Google Scholar, 11.Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (442) Google Scholar). results that mAKAPβ serves an additional in the regulation of myocyte hypertrophy through the of AC5 activity by PKA feedback using containing AC5 and mAKAPβ performed in the of and phosphodiesterase revealed the importance of PKA associated with the complex to AC5 inhibition (Fig. Accordingly, expression in myocytes of the mAKAPβ endogenous complexes and increased total cAMP, in increased and protein in the absence of stimulation. of binding also AC5 from in the which cAMP In (Fig. mAKAPβ is to cAMP and Thus, of the mAKAPβ complex cAMP signaling to cAMP while of cAMP in to receptor stimulation. In addition to a of cAMP results also a for the of cAMP levels in of AC5 be to the heart, of AC5 activity in the of cardiac 1999; PubMed Scopus Google Scholar). The results obtained by mAKAPβ a by which to cAMP by the of ACs with In we show for the that the anchoring of AC to an complex is required for the control of cAMP-dependent a for localized cAMP signaling and a in cAMP signaling and We for and and Kirk for AC5 wild type and hearts. with