cAMP-stimulated Protein Phosphatase 2A Activity Associated with Muscle A Kinase-anchoring Protein (mAKAP) Signaling Complexes Inhibits the Phosphorylation and Activity of the cAMP-specific Phosphodiesterase PDE4D3

Abstract

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085–2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56δ. Like PDE4D3, B56δ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56δ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56δ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56δ phosphorylation. In general, PKA·PP2A·mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085–2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56δ. Like PDE4D3, B56δ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56δ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56δ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56δ phosphorylation. In general, PKA·PP2A·mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. IntroductioncAMP controls a plethora of processes in virtually every cell type, including gene expression, intermediary metabolism, and ion channel activity. In most cases, cAMP is produced through G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorPDE4D3phosphodiesterase 4D3mAKAPmuscle A kinase-anchoring proteinPKAprotein kinase AMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinasePP1protein phosphatase 1PP2Aprotein phosphatase 2APP2Bprotein phosphatase 2BGFPgreen fluorescent proteinVSVvesicular stomatitis virusGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acidOAokadaic acidFskforskolinCPT-cAMP8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphatePKIprotein kinase inhibitorIBMX3-isobutyl-1-methylxanthine. activation of the stimulatory Gα protein (Gαs), which in turn activates adenylyl cyclase that catalyzes the conversion of ATP to cAMP. In the heart, a variety of GPCRs promote the production of this small diffusible second messenger. However, a long standing finding is that the cellular response to cAMP signaling varies in the myocyte depending upon the upstream GPCR (1Hayes J.S. Brunton L.L. Mayer S.E. J. Biol. Chem. 1980; 255: 5113-5119Abstract Full Text PDF PubMed Google Scholar, 2Brunton L.L. Hayes J.S. Mayer S.E. Nature. 1979; 280: 78-80Crossref PubMed Scopus (67) Google Scholar, 3Keely S.L. Res. Commun. Chem. Pathol. Pharmacol. 1977; 18: 283-290PubMed Google Scholar, 4Keely S.L. Mol. Pharmacol. 1979; 15: 235-245PubMed Google Scholar). This observation has led to the hypothesis that cAMP production in response to the stimulation of individual GPCRs is restricted to discrete subcellular domains, conferring spatiotemporal control of cAMP production as well as response specificity (5Steinberg S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (319) Google Scholar, 6Dodge-Kafka K.L. Langeberg L. Scott J.D. Circ. Res. 2006; 98: 993-1001Crossref PubMed Scopus (104) Google Scholar). Understanding the mechanisms that confine cAMP to these microdomains remains of considerable interest.Recent experimentation involving live cell imaging of cardiac myocytes has revealed that type 4 phosphodiesterases (PDE4) are important for the spatiotemporal control of cAMP following β-adrenergic receptor stimulation (7Fischmeister R. Castro L.R. Abi-Gerges A. Rochais F. Jurevicius J. Leroy J. Vandecasteele G. Circ. Res. 2006; 99: 816-828Crossref PubMed Scopus (301) Google Scholar). Individual PDE4 isoforms may contribute to the local regulation of cAMP in different cellular compartments. For example, we have shown that the alternatively spliced PDE4 isoform D3 (PDE4D3) is bound by the scaffold protein mAKAP (muscle A kinase-anchoring protein) in cardiac myocytes (8Dodge-Kafka K.L. Kapiloff M.S. Eur. J. Cell Biol. 2006; 85: 593-602Crossref PubMed Scopus (58) Google Scholar, 9Dodge-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 (435) Google Scholar, 10Dodge 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 (398) Google Scholar). Because mAKAP is tethered to the nuclear envelope by nesprin-1α (11Pare G.C. Easlick J.L. Mislow J.M. McNally E.M. Kapiloff M.S. Exp. Cell Res. 2005; 303: 388-399Crossref PubMed Scopus (90) Google Scholar), PDE4D3-bound mAKAP likely controls perinuclear cAMP levels. Signaling through mAKAP multimolecular signaling complexes has been implicated in the regulation of myocyte hypertrophy and in the regulation of gene expression during hypoxia (9Dodge-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 (435) Google Scholar, 12Pare 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 (105) Google Scholar, 13Wong W. Goehring A.S. Kapiloff M.S. Langeberg L.K. Scott J.D. Sci. Signal. 2008; 1: ra18Crossref PubMed Scopus (47) Google Scholar, 14Li J. Negro A. Lopez J. Bauman A.L. Henson E. Dodge-Kafka K. Kapiloff M.S. J. Mol. Cell. Cardiol. 2010; 48: 387-394Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As its name implies, mAKAP binds the cAMP-dependent protein kinase PKA, and PKA binding to mAKAP is required for PDE4D3 phosphorylation. In response to elevated cAMP levels, mAKAP-bound PKA phosphorylates PDE4D3 on serine residues 13 and 54, resulting in 2–3-fold increased PDE4D3 binding to the complex and PDE activity (10Dodge 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 (398) Google Scholar, 15Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 16Carlisle 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 (94) Google Scholar). Because increased PDE4D3 activity accelerates cAMP degradation, PKA and PDE4D3 constitute a negative feedback loop that modulates both local cAMP levels and PKA activity (10Dodge 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 (398) Google Scholar). PDE4D3 bound to mAKAP serves not only as a PDE, but also as an adapter protein, recruiting the MAPKs MEK5 and ERK5 and the cAMP-dependent Rap1-guanine nucleotide exchange factor Epac1 to mAKAP complexes (9Dodge-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 (435) Google Scholar). Activation of MEK5 and ERK5 by upstream signals results in PDE4D3 phosphorylation on serine residue 579, inhibiting the PDE and promoting cAMP accumulation and PKA activation (9Dodge-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 (435) Google Scholar, 17MacKenzie S.J. Baillie G.S. McPhee I. Bolger G.B. Houslay M.D. J. Biol. Chem. 2000; 275: 16609-16617Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 18Hoffmann R. Baillie G.S. MacKenzie S.J. Yarwood S.J. Houslay M.D. EMBO J. 1999; 18: 893-903Crossref PubMed Scopus (224) Google Scholar). Elevated cAMP levels will also activate mAKAP-associated Epac1. However, through Rap1, Epac1 inhibits ERK5 activity, preventing PDE4D3 inhibition (9Dodge-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 (435) Google Scholar). As a result, Epac1, ERK5, and PDE4D3 constitute a second negative feedback loop that will attenuate cAMP levels in the vicinity of mAKAP complexes. Importantly, both of these negative feedback loops intrinsic to the mAKAP signaling complex depend upon the regulation of PDE4D3 activity as the key element controlling local cAMP levels.Although we have described how mAKAP-bound PDE4D3 may be regulated by PKA and ERK5 phosphorylation, the phosphatase(s) involved in PDE4D3 dephosphorylation have remained unknown. We now show that PP2A bound to the mAKAP complex catalyzes the dephosphorylation of PDE4D3 Ser-54, inhibiting the PDE. Although previously thought to be a constitutive, housekeeping enzyme, it has become apparent that PP2A contributes to the regulation of many phosphorylation events. For example, in the cardiac myocyte, PP2A is involved in the modulation of calcium and MAPK signaling (19duBell W.H. Lederer W.J. Rogers T.B. J. Physiol. 1996; 493: 793-800Crossref PubMed Scopus (79) Google Scholar, 20duBell W.H. Gigena M.S. Guatimosim S. Long X. Lederer W.J. Rogers T.B. Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H38-H48Crossref PubMed Scopus (55) Google Scholar, 21Liu Q. Hofmann P.A. Am. J. Physiol. Heart Circ. Physiol. 2004; 286: H2204-H2212Crossref PubMed Scopus (140) Google Scholar). The current challenge in the study of this phosphatase is to understand its spatiotemporal regulation. PP2A is a serine/threonine phosphatase that exists as a heterotrimeric complex consisting of a stable, ubiquitously expressed catalytic (PP2A-C) and scaffolding (PP2A-A) subunit heterodimer and one of 21 known divergent B subunits (22Lechward K. Awotunde O.S. Swiatek W. Muszyñska G. Acta Biochim. Pol. 2001; 48: 921-933Crossref PubMed Scopus (171) Google Scholar, 23Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (597) Google Scholar). PP2A-B subunits are grouped into three unrelated families termed B (or PR55), B′ (or B56) and B″ (or PR72) and are proposed to regulate both the catalytic activity and the intracellular targeting of the phosphatase (24Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar). As now revealed, PP2A associated with mAKAP complexes contain B56δ B subunits. Recently published work demonstrates that B56δ is a PKA substrate, and its phosphorylation enhances PP2A catalytic activity (25Ahn J.H. McAvoy T. Rakhilin S.V. Nishi A. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2979-2984Crossref PubMed Scopus (209) Google Scholar). Accordingly, we show that phosphorylation of B56δ by mAKAP-bound PKA increases PDE4D3 dephosphorylation. Our results provide the mechanistic details of mAKAP-anchored PP2A regulation of PDE4D3, elucidating further how mAKAP signaling complexes may regulate a discrete intracellular cAMP signaling domain through a set of interlacing positive and negative feedback loops.DISCUSSIONThe results described in this paper define the biochemical mechanism for the dephosphorylation and inactivation of PKA-phosphorylated PDE4D3 bound by the scaffold protein mAKAP. We discovered that a PP2A heterotrimer composed of A, C, and B56δ subunits binds a C-terminal site on mAKAP distinct from the binding for known mAKAP The of PP2A with the mAKAP scaffold is of in important and by binding both PP2A and PDE4D3, mAKAP the phosphatase in to the PDE, for PDE4D3 dephosphorylation and by binding both PKA and mAKAP promotes cAMP-dependent phosphorylation of the PP2A B56δ subunit and of PP2A activity The of multimolecular signaling complex upon expression of mAKAP binding for PP2A and of phosphatase targeting to specificity proposed in the with the of the protein as the subunit A.L. Scott J.D. Cell Biol. 2002; PubMed Scopus Google Scholar). Because of this phosphatase targeting have been (24Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar). an important mechanism to phosphatases with and protein We have published that mAKAP binds and that this is important for activation in myocytes J. Negro A. Lopez J. Bauman A.L. Henson E. Dodge-Kafka K. Kapiloff M.S. J. Mol. Cell. Cardiol. 2010; 48: 387-394Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, binding to mAKAP not to regulate PDE4D3 inhibition of not PDE4D3 phosphorylation PDE activity Our a for PP2A bound to mAKAP in dephosphorylation of the PDE as a result, in the control of local cAMP of phosphatases in cellular cAMP concentration has to be In PP2A to regulate both activity and phosphorylation S. A. S. E. Biochem. J. 1999; PubMed Scopus (54) Google Scholar). In to phosphorylated by PKA on Ser-54, PDE4D3 is phosphorylated on by including by ERK5 in mAKAP complexes (9Dodge-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 (435) Google Scholar, 18Hoffmann R. Baillie G.S. MacKenzie S.J. Yarwood S.J. Houslay M.D. EMBO J. 1999; 18: 893-903Crossref PubMed Scopus (224) Google Scholar). Although not to mAKAP and may PDE4D3 in cellular the of to PDE4D3 phosphorylation this work will be required to the phosphatase(s) responsible for the dephosphorylation of mAKAP-bound PDE4D3 as well as the second PKA site on PDE4D3, 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 (94) Google hypothesis that to the of PKA by both to the signaling complex. In this we demonstrate a for PKA in the mAKAP the PP2A B56δ work that phosphorylation of B56δ PP2A activity and dephosphorylation of (25Ahn J.H. McAvoy T. Rakhilin S.V. Nishi A. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2979-2984Crossref PubMed Scopus (209) Google Scholar). In with our stimulation of cardiac myocytes with β-adrenergic receptor increases PP2A activity Mol. Pharmacol. 2008; PubMed Scopus Google Scholar). We that the mAKAP scaffold may this the of the protein with both PKA and PP2A is important for the in phosphatase activity one a for mAKAP in the regulation of phosphatase activity in the upon our we a in which PP2A serves a in cAMP levels mAKAP signaling complexes PP2A in mAKAP complexes PDE4D3 in a in the of GPCR stimulation for a in cAMP levels in response to following of cAMP levels by GPCR PKA will both PDE4D3 and PP2A In to the negative feedback on cAMP levels by PDE4D3 phosphorylation, PKA phosphorylation of PP2A PDE4D3 inhibiting PDE4D3 phosphorylation, PP2A and the of local cAMP as of a positive feedback in with the inhibition of PDE4D3 by mAKAP-bound ERK5 that we have previously described not (9Dodge-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 (435) Google Scholar), the mAKAP signaling complex is to regulate local cAMP levels both by feedback loops intrinsic to the complex as well as by with upstream MAPK signaling these signaling that on PDE4D3 regulate the of cAMP in myocytes by different of upstream for example, and will further For example, it has been that PP2A expression and intracellular are in heart S. M. J. K.L. A. E. J. 2001; 104: PubMed Scopus Google Scholar, X. Circ. Res. 2005; PubMed Scopus Google Scholar). positive feedback negative feedback controls cAMP levels local to mAKAP complexes may depend both on the of PP2A binding to mAKAP and the of PDE4D3 phosphorylation and dephosphorylation by PKA and PP2A in we have discovered a mechanism by which the scaffold protein mAKAP regulation of PDE4D3 activity through the with PDE4D3, PKA, and PP2A. of the three is likely to an important in the control of cAMP concentration in the vicinity of perinuclear mAKAP complex. This regulation of local cAMP by the mAKAP likely a for and phosphatase in the control of cAMP IntroductioncAMP controls a plethora of processes in virtually every cell type, including gene expression, intermediary metabolism, and ion channel activity. In most cases, cAMP is produced through G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorPDE4D3phosphodiesterase 4D3mAKAPmuscle A kinase-anchoring proteinPKAprotein kinase AMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinasePP1protein phosphatase 1PP2Aprotein phosphatase 2APP2Bprotein phosphatase 2BGFPgreen fluorescent proteinVSVvesicular stomatitis virusGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acidOAokadaic acidFskforskolinCPT-cAMP8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphatePKIprotein kinase inhibitorIBMX3-isobutyl-1-methylxanthine. activation of the stimulatory Gα protein (Gαs), which in turn activates adenylyl cyclase that catalyzes the conversion of ATP to cAMP. In the heart, a variety of GPCRs promote the production of this small diffusible second messenger. However, a long standing finding is that the cellular response to cAMP signaling varies in the myocyte depending upon the upstream GPCR (1Hayes J.S. Brunton L.L. Mayer S.E. J. Biol. Chem. 1980; 255: 5113-5119Abstract Full Text PDF PubMed Google Scholar, 2Brunton L.L. Hayes J.S. Mayer S.E. Nature. 1979; 280: 78-80Crossref PubMed Scopus (67) Google Scholar, 3Keely S.L. Res. Commun. Chem. Pathol. Pharmacol. 1977; 18: 283-290PubMed Google Scholar, 4Keely S.L. Mol. Pharmacol. 1979; 15: 235-245PubMed Google Scholar). This observation has led to the hypothesis that cAMP production in response to the stimulation of individual GPCRs is restricted to discrete subcellular domains, conferring spatiotemporal control of cAMP production as well as response specificity (5Steinberg S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (319) Google Scholar, 6Dodge-Kafka K.L. Langeberg L. Scott J.D. Circ. Res. 2006; 98: 993-1001Crossref PubMed Scopus (104) Google Scholar). Understanding the mechanisms that confine cAMP to these microdomains remains of considerable interest.Recent experimentation involving live cell imaging of cardiac myocytes has revealed that type 4 phosphodiesterases (PDE4) are important for the spatiotemporal control of cAMP following β-adrenergic receptor stimulation (7Fischmeister R. Castro L.R. Abi-Gerges A. Rochais F. Jurevicius J. Leroy J. Vandecasteele G. Circ. Res. 2006; 99: 816-828Crossref PubMed Scopus (301) Google Scholar). Individual PDE4 isoforms may contribute to the local regulation of cAMP in different cellular compartments. For example, we have shown that the alternatively spliced PDE4 isoform D3 (PDE4D3) is bound by the scaffold protein mAKAP (muscle A kinase-anchoring protein) in cardiac myocytes (8Dodge-Kafka K.L. Kapiloff M.S. Eur. J. Cell Biol. 2006; 85: 593-602Crossref PubMed Scopus (58) Google Scholar, 9Dodge-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 (435) Google Scholar, 10Dodge 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 (398) Google Scholar). Because mAKAP is tethered to the nuclear envelope by nesprin-1α (11Pare G.C. Easlick J.L. Mislow J.M. McNally E.M. Kapiloff M.S. Exp. Cell Res. 2005; 303: 388-399Crossref PubMed Scopus (90) Google Scholar), PDE4D3-bound mAKAP likely controls perinuclear cAMP levels. Signaling through mAKAP multimolecular signaling complexes has been implicated in the regulation of myocyte hypertrophy and in the regulation of gene expression during hypoxia (9Dodge-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 (435) Google Scholar, 12Pare 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 (105) Google Scholar, 13Wong W. Goehring A.S. Kapiloff M.S. Langeberg L.K. Scott J.D. Sci. Signal. 2008; 1: ra18Crossref PubMed Scopus (47) Google Scholar, 14Li J. Negro A. Lopez J. Bauman A.L. Henson E. Dodge-Kafka K. Kapiloff M.S. J. Mol. Cell. Cardiol. 2010; 48: 387-394Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As its name implies, mAKAP binds the cAMP-dependent protein kinase PKA, and PKA binding to mAKAP is required for PDE4D3 phosphorylation. In response to elevated cAMP levels, mAKAP-bound PKA phosphorylates PDE4D3 on serine residues 13 and 54, resulting in 2–3-fold increased PDE4D3 binding to the complex and PDE activity (10Dodge 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 (398) Google Scholar, 15Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 16Carlisle 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 (94) Google Scholar). Because increased PDE4D3 activity accelerates cAMP degradation, PKA and PDE4D3 constitute a negative feedback loop that modulates both local cAMP levels and PKA activity (10Dodge 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 (398) Google Scholar). PDE4D3 bound to mAKAP serves not only as a PDE, but also as an adapter protein, recruiting the MAPKs MEK5 and ERK5 and the cAMP-dependent Rap1-guanine nucleotide exchange factor Epac1 to mAKAP complexes (9Dodge-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 (435) Google Scholar). Activation of MEK5 and ERK5 by upstream signals results in PDE4D3 phosphorylation on serine residue 579, inhibiting the PDE and promoting cAMP accumulation and PKA activation (9Dodge-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 (435) Google Scholar, 17MacKenzie S.J. Baillie G.S. McPhee I. Bolger G.B. Houslay M.D. J. Biol. Chem. 2000; 275: 16609-16617Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 18Hoffmann R. Baillie G.S. MacKenzie S.J. Yarwood S.J. Houslay M.D. EMBO J. 1999; 18: 893-903Crossref PubMed Scopus (224) Google Scholar). Elevated cAMP levels will also activate mAKAP-associated Epac1. However, through Rap1, Epac1 inhibits ERK5 activity, preventing PDE4D3 inhibition (9Dodge-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 (435) Google Scholar). As a result, Epac1, ERK5, and PDE4D3 constitute a second negative feedback loop that will attenuate cAMP levels in the vicinity of mAKAP complexes. Importantly, both of these negative feedback loops intrinsic to the mAKAP signaling complex depend upon the regulation of PDE4D3 activity as the key element controlling local cAMP levels.Although we have described how mAKAP-bound PDE4D3 may be regulated by PKA and ERK5 phosphorylation, the phosphatase(s) involved in PDE4D3 dephosphorylation have remained unknown. We now show that PP2A bound to the mAKAP complex catalyzes the dephosphorylation of PDE4D3 Ser-54, inhibiting the PDE. Although previously thought to be a constitutive, housekeeping enzyme, it has become apparent that PP2A contributes to the regulation of many phosphorylation events. For example, in the cardiac myocyte, PP2A is involved in the modulation of calcium and MAPK signaling (19duBell W.H. Lederer W.J. Rogers T.B. J. Physiol. 1996; 493: 793-800Crossref PubMed Scopus (79) Google Scholar, 20duBell W.H. Gigena M.S. Guatimosim S. Long X. Lederer W.J. Rogers T.B. Am. J. Physiol. Heart Circ. Physiol. 2002; 282: H38-H48Crossref PubMed Scopus (55) Google Scholar, 21Liu Q. Hofmann P.A. Am. J. Physiol. Heart Circ. Physiol. 2004; 286: H2204-H2212Crossref PubMed Scopus (140) Google Scholar). The current challenge in the study of this phosphatase is to understand its spatiotemporal regulation. PP2A is a serine/threonine phosphatase that exists as a heterotrimeric complex consisting of a stable, ubiquitously expressed catalytic (PP2A-C) and scaffolding (PP2A-A) subunit heterodimer and one of 21 known divergent B subunits (22Lechward K. Awotunde O.S. Swiatek W. Muszyñska G. Acta Biochim. Pol. 2001; 48: 921-933Crossref PubMed Scopus (171) Google Scholar, 23Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (597) Google Scholar). PP2A-B subunits are grouped into three unrelated families termed B (or PR55), B′ (or B56) and B″ (or PR72) and are proposed to regulate both the catalytic activity and the intracellular targeting of the phosphatase (24Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar). As now revealed, PP2A associated with mAKAP complexes contain B56δ B subunits. Recently published work demonstrates that B56δ is a PKA substrate, and its phosphorylation enhances PP2A catalytic activity (25Ahn J.H. McAvoy T. Rakhilin S.V. Nishi A. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2979-2984Crossref PubMed Scopus (209) Google Scholar). Accordingly, we show that phosphorylation of B56δ by mAKAP-bound PKA increases PDE4D3 dephosphorylation. Our results provide the mechanistic details of mAKAP-anchored PP2A regulation of PDE4D3, elucidating further how mAKAP signaling complexes may regulate a discrete intracellular cAMP signaling domain through a set of interlacing positive and negative feedback