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
2010; Elsevier BV; Volume: 285; Issue: 15 Linguagem: Inglês
10.1074/jbc.m109.034868
ISSN1083-351X
AutoresKimberly L. Dodge‐Kafka, Andrea L. Bauman, Nicole Mayer, Edward Henson, Lorena Heredia, Jung‐Hyuck Ahn, Thomas McAvoy, Angus C. Nairn, Michael S. Kapiloff,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoThe 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 sites for other known mAKAP partners (Fig. 3). The association of PP2A with the mAKAP scaffold is of functional significance in two important and novel ways. First, by binding both PP2A and PDE4D3, mAKAP sequesters the phosphatase in close proximity to the PDE, allowing for efficient PDE4D3 dephosphorylation and down-regulation (Fig. 4). Second, by binding both PKA and PP2A, mAKAP promotes cAMP-dependent phosphorylation of the PP2A B56δ subunit and induction of PP2A activity (Fig. 6). The relevance of multimolecular signaling complex formation was evident upon expression of mAKAP mutants lacking binding sites for PP2A and PKA.The concept of phosphatase targeting to generate substrate specificity was first proposed in the mid-1980s with the identification of the glycogen particle-associated protein as the first PP1-targeting subunit (34Bauman A.L. Scott J.D. Nat. Cell Biol. 2002; 4: E203-E206Crossref PubMed Scopus (114) Google Scholar). Because of this initial observation, several other phosphatase targeting motifs have been determined (24Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar). AKAPs represent an important mechanism to link phosphatases with their appropriate substrates, and several AKAPs bind protein phosphatases. We have recently published that mAKAP binds PP2B and that this interaction is important for PP2B-dependent NFATc3 activation in myocytes (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). However, PP2B binding to mAKAP does not appear to regulate PDE4D3 because inhibition of PP2B did not affect PDE4D3 Ser-54 phosphorylation or PDE activity (Fig. 1). Our data support a unique role for PP2A bound to mAKAP in dephosphorylation of the PDE and, as a result, in the control of local cAMP levels.The overall role of phosphatases in regulating cellular cAMP concentration has yet to be fully explored. In rat adipocytes, PP2A was found to regulate both PDE3B activity and phosphorylation (35Resjo S. Oknianska A. Zolnierowicz S. Manganiello V. Degerman E. Biochem. J. 1999; 341: 839-845Crossref PubMed Scopus (54) Google Scholar). In addition to being phosphorylated by PKA on Ser-54, PDE4D3 is phosphorylated on Ser-579 by MAPKs, including by ERK5 present 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 PP1 does not appear to bind mAKAP (Fig. 2 and supplemental Fig. 3), PP1 may dephosphorylate PDE4D3 Ser-579 in other cellular domains because the addition of purified PP1 to isolated PDE4D3 decreased phosphorylation at this site. Further work will be required to identify the phosphatase(s) responsible for the dephosphorylation of mAKAP-bound PDE4D3 at Ser-579 as well as the second PKA site on PDE4D3, Ser-16 (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).The anchoring hypothesis suggests that AKAPs function to target the actions of PKA toward specific substrates by localizing both proteins to the same signaling complex. In this report, we demonstrate a new target for PKA in the mAKAP complex, the PP2A B56δ subunit. Previous work found that phosphorylation of B56δ stimulated PP2A activity and enhanced dephosphorylation of DARPP-32 (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 accordance with our results, stimulation of cardiac myocytes with β-adrenergic receptor agonists increases PP2A activity (36De Arcangelis V. Soto D. Xiang Y. Mol. Pharmacol. 2008; 74: 1453-1462Crossref PubMed Scopus (29) Google Scholar). We propose that the mAKAP scaffold may facilitate this event because the association of the anchoring protein with both PKA and PP2A is important for the cAMP-enhanced increase in phosphatase activity (FIGURE 4, FIGURE 5, FIGURE 6). Hence, one can imagine a role for mAKAP in the regulation of phosphatase activity in the heart.Based upon our results, we propose a model in which PP2A serves a dual role in regulating cAMP levels near mAKAP signaling complexes (Fig. 8). First, PP2A in mAKAP complexes should maintain PDE4D3 in a dephosphorylated, minimally active state in the absence of GPCR stimulation (Fig. 8A), presumably allowing for a more rapid rise in cAMP levels in response to agonist. Second, following induction of activating cAMP levels by GPCR stimulation, PKA will phosphorylate both PDE4D3 and PP2A (Fig. 8B). In contrast to the negative feedback on cAMP levels mediated by enhanced PDE4D3 phosphorylation, PKA phosphorylation of PP2A opposes PDE4D3 activation. By inhibiting PDE4D3 phosphorylation, PP2A presumably potentiates and prolongs the actions of local cAMP as part of a positive feedback loop. Thus, in conjunction with the potential inhibition of PDE4D3 by mAKAP-bound ERK5 that we have previously described (data not shown) (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 poised to finely regulate local cAMP levels both by multiple feedback loops intrinsic to the complex as well as by cross-talk with upstream MAPK signaling pathways. How these multiple signaling pathways that converge on PDE4D3 ultimately regulate the kinetics of cAMP metabolism in myocytes stimulated by different combinations of upstream signals, including, for example, adrenergic and cytokine receptors, will require further investigation. For example, it has been observed that PP2A expression and intracellular localization are altered in heart failure (37Reiken S. Gaburjakova M. Gaburjakova J. He Kl K.L. Prieto A. Becker E. Yi Gh G.H. Wang J. Burkhoff D. Marks A.R. Circulation. 2001; 104: 2843-2848Crossref PubMed Scopus (151) Google Scholar, 38Ai X. Pogwizd S.M. Circ. Res. 2005; 96: 54-63Crossref PubMed Scopus (172) Google Scholar). Whether PP2A-mediated positive feedback or PDE4D3-mediated negative feedback predominately controls cAMP levels local to mAKAP complexes may ultimately depend both on the stoichiometry of PP2A binding to mAKAP and the relative rates of PDE4D3 phosphorylation and dephosphorylation by PKA and PP2A in disease states.In conclusion, we have discovered a novel mechanism by which the scaffold protein mAKAP maintains dynamic regulation of anchored PDE4D3 activity through the association with PDE4D3, PKA, and PP2A. Each of the three enzymes is likely to play an important role in the temporal control of cAMP concentration in the vicinity of perinuclear mAKAP complex. This intricate regulation of local cAMP by the mAKAP "signalosome" likely represents a broader role for AKAPs and phosphatase in the control of cAMP compartmentation. 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. 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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.
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