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• W2080351377 startingPage "16170" @default.
• W2080351377 abstract "β-Arrestin plays a key role in regulating β2-adrenoreceptor signaling by interdicting activation of adenylyl cyclase and selectively sequestering cAMP phosphodiesterase-4D5 (PDE4D5) for delivery of an active cAMP degrading system to the site of cAMP synthesis. Here we show that the β-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing β2-adrenoceptors. Reconstitution analyses in β-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a β-arrestin-scaffolded pool of the E3-ubiquitin ligase, Mdm2, mediates PDE4D5 ubiquitination. Critical for this is the ubiquitin-interacting motif located in the extreme C terminus of PDE4D5, which is specific to the PDE4D sub-family. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquitination occurs at Lys-48, Lys-53, and Lys-78, which are located within its isoform-specific N-terminal region, as well as at Lys-140 located within its regulatory UCR1 module. We suggest that mono-ubiquitination at Lys-140 primes PDE4D5 for a subsequent cascade of polyubiquitination occurring within its isoform-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated β-arrestin sub-population that is likely to be protected from Mdm2-mediated ubiquitination due to steric hindrance caused by sequestered PDE4D5. Ubiquitination of PDE4D5 elicits an increase in the fraction of PDE4D5 sequestered by β-arrestin in cells, thereby contributing to the fidelity of PDE4D5-β-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1. β-Arrestin plays a key role in regulating β2-adrenoreceptor signaling by interdicting activation of adenylyl cyclase and selectively sequestering cAMP phosphodiesterase-4D5 (PDE4D5) for delivery of an active cAMP degrading system to the site of cAMP synthesis. Here we show that the β-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing β2-adrenoceptors. Reconstitution analyses in β-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a β-arrestin-scaffolded pool of the E3-ubiquitin ligase, Mdm2, mediates PDE4D5 ubiquitination. Critical for this is the ubiquitin-interacting motif located in the extreme C terminus of PDE4D5, which is specific to the PDE4D sub-family. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquitination occurs at Lys-48, Lys-53, and Lys-78, which are located within its isoform-specific N-terminal region, as well as at Lys-140 located within its regulatory UCR1 module. We suggest that mono-ubiquitination at Lys-140 primes PDE4D5 for a subsequent cascade of polyubiquitination occurring within its isoform-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated β-arrestin sub-population that is likely to be protected from Mdm2-mediated ubiquitination due to steric hindrance caused by sequestered PDE4D5. Ubiquitination of PDE4D5 elicits an increase in the fraction of PDE4D5 sequestered by β-arrestin in cells, thereby contributing to the fidelity of PDE4D5-β-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1. Mdm2 directs the ubiquitination of Β-arrestin-sequestered cAMP phosphodiesterase-4D5.Journal of Biological ChemistryVol. 284Issue 32PreviewVOLUME 284 (2009) PAGES 16170–16182 Full-Text PDF Open AccessWithdrawal: Mdm2 directs the ubiquitination of β-arrestin-sequestered cAMP phosphodiesterase-4D5Journal of Biological ChemistryVol. 295Issue 34PreviewVOLUME 284 (2009) PAGES 16170–16182 Full-Text PDF Open Access The second messenger cAMP controls key biological processes in all mammalian cells (1Beavo J.A. Brunton L.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 710-718Crossref PubMed Scopus (725) Google Scholar, 2Taskén K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (625) Google Scholar, 3Taylor S.S. Kim C. 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In defining compartmentalized cAMP signaling, members of the phosphodiesterase-4 (PDE4) 3The abbreviations used are: PDE4cAMP specific phosphodiesterase-4UIMubiquitin-interacting motifUCR1upstream conserved region 1UCR2upstream conserved region 2AKAPprotein kinase A-anchoring proteinRACK1receptor for activated protein kinase CDISC1disrupted in schizophrenia 1β2ARβ2adrenoceptorGPCRG-protein-coupled receptorUbubiquitinE1 ligaseUb-activating ligase 1E2 ligaseUb-conjugating ligase 2E3 ligaseUb-conjugating ligase 3Mdm2murine double minute 2MEFmouse embryo fibroblastaaamino acid(s)VSVvesicular stomatitis virus. family (5Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (955) Google Scholar, 9Houslay M.D. Schafer P. Zhang K.Y. Drug Discov. Today. 2005; 10: 1503-1519Crossref PubMed Scopus (561) Google Scholar), which specifically hydrolyze cAMP, play a pivotal role in many cell types (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 11Terrin A. Di Benedetto G. Pertegato V. Cheung Y.F. Baillie G. Lynch M.J. Elvassore N. Prinz A. Herberg F.W. Houslay M.D. Zaccolo M. J. Cell Biol. 2006; 175: 441-451Crossref PubMed Scopus (149) Google Scholar, 12Willoughby D. Baillie G.S. Lynch M.J. Ciruela A. Houslay M.D. Cooper D.M. J. Biol. Chem. 2007; 282: 34235-34249Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 13Baillie G.S. Scott J.D. Houslay M.D. FEBS Lett. 2005; 579: 3264-3270Crossref PubMed Scopus (181) Google Scholar). Alternative mRNA splicing of transcripts from four genes (PDE4A/B/C/D) generates over 20 distinct PDE4 isoforms, each characterized by a unique N-terminal region involved in targeting the isoform to specific signaling complexes (7Houslay M.D. Adams D.R. Biochem. J. 2003; 370: 1-18Crossref PubMed Scopus (648) Google Scholar, 9Houslay M.D. Schafer P. Zhang K.Y. Drug Discov. Today. 2005; 10: 1503-1519Crossref PubMed Scopus (561) Google Scholar, 14Baillie G.S. Houslay M.D. Curr. Opin. Cell Biol. 2005; 17: 129-134Crossref PubMed Scopus (116) Google Scholar). PDE4-selective inhibitors provide potent anti-inflammatory therapeutics targeted to inflammatory pulmonary disease (9Houslay M.D. Schafer P. Zhang K.Y. Drug Discov. Today. 2005; 10: 1503-1519Crossref PubMed Scopus (561) Google Scholar, 15Huang Z. Mancini J.A. Curr. Med. Chem. 2006; 13: 3253-3262Crossref PubMed Scopus (73) Google Scholar) and depression (16O'Donnell J.M. Zhang H.T. Trends Pharmacol. Sci. 2004; 25: 158-163Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), with the PDE4D gene being linked to stroke (17Nakayama T. Asai S. Sato N. Soma M. Curr. Med. Chem. 2007; 14: 3171-3178Crossref PubMed Scopus (12) Google Scholar) and the PDE4B gene to schizophrenia (18Millar J.K. Pickard B.S. Mackie S. James R. Christie S. Buchanan S.R. Malloy M.P. Chubb J.E. Huston E. Baillie G.S. Thomson P.A. Hill E.V. Brandon N.J. Rain J.C. Camargo L.M. Whiting P.J. Houslay M.D. Blackwood D.H. Muir W.J. Porteous D.J. Science. 2005; 310: 1187-1191Crossref PubMed Scopus (568) Google Scholar). cAMP specific phosphodiesterase-4 ubiquitin-interacting motif upstream conserved region 1 upstream conserved region 2 protein kinase A-anchoring protein receptor for activated protein kinase C disrupted in schizophrenia 1 β2adrenoceptor G-protein-coupled receptor ubiquitin Ub-activating ligase 1 Ub-conjugating ligase 2 Ub-conjugating ligase 3 murine double minute 2 mouse embryo fibroblast amino acid(s) vesicular stomatitis virus. Specific PDE4 isoforms and sub-families have distinct, non-redundant phenotypic roles identified by dominant negative approaches, small interference RNA-mediated knockdown (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 11Terrin A. Di Benedetto G. Pertegato V. Cheung Y.F. Baillie G. Lynch M.J. Elvassore N. Prinz A. Herberg F.W. Houslay M.D. Zaccolo M. J. Cell Biol. 2006; 175: 441-451Crossref PubMed Scopus (149) Google Scholar, 12Willoughby D. Baillie G.S. Lynch M.J. Ciruela A. Houslay M.D. Cooper D.M. J. Biol. Chem. 2007; 282: 34235-34249Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (304) Google Scholar, 20McCahill A. McSorley T. Huston E. Hill E.V. Lynch M.J. Gall I. Keryer G. Lygren B. Tasken K. van Heeke G. Houslay M.D. Cell. Signal. 2005; 17: 1158-1173Crossref PubMed Scopus (98) Google Scholar) and genetic ablation (21Ariga M. Neitzert B. Nakae S. Mottin G. Bertrand C. Pruniaux M.P. Jin S.L. Conti M. J. Immunol. 2004; 173: 7531-7538Crossref PubMed Scopus (149) Google Scholar, 22Jin S.L. Lan L. Zoudilova M. Conti M. J. Immunol. 2005; 175: 1523-1531Crossref PubMed Scopus (195) Google Scholar). Pivotal to this is the recruitment of particular PDE4 isoforms to specific signaling complexes and intracellular locales, which confers a spatial dimension on cAMP signaling (14Baillie G.S. Houslay M.D. Curr. Opin. Cell Biol. 2005; 17: 129-134Crossref PubMed Scopus (116) Google Scholar). Indeed, particular PDE4 isoforms have been shown to interact with the signaling scaffold proteins β-arrestin (23Perry S.J. Baillie G.S. Kohout T.A. McPhee I. Magiera M.M. Ang K.L. Miller W.E. McLean A.J. Conti M. Houslay M.D. Lefkowitz R.J. Science. 2002; 298: 834-836Crossref PubMed Scopus (410) Google Scholar), RACK1 (24Smith K.J. Baillie G.S. Hyde E.I. Li X. Houslay T.M. McCahill A. Dunlop A.J. Bolger G.B. Klussmann E. Adams D.R. Houslay M.D. Cell. Signal. 2007; 19: 2612-2624Crossref PubMed Scopus (48) Google Scholar), AKAPs (25Dodge 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 (401) Google Scholar, 26Taskén K.A. Collas P. Kemmner W.A. Witczak O. Conti M. Taskén K. J. Biol. Chem. 2001; 276: 21999-22002Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 27Stefan E. Wiesner B. Baillie G.S. Mollajew R. Henn V. Lorenz D. Furkert J. Santamaria K. Nedvetsky P. Hundsrucker C. Beyermann M. Krause E. Pohl P. Gall I. MacIntyre A.N. Bachmann S. Houslay M.D. Rosenthal W. Klussmann E. J. Am. Soc. Nephrol. 2007; 18: 199-212Crossref PubMed Scopus (117) Google Scholar), and DISC1 (28Murdoch H. Mackie S. Collins D.M. Hill E.V. Bolger G.B. Klussmann E. Porteous D.J. Millar J.K. Houslay M.D. J. Neurosci. 2007; 27: 9513-9524Crossref PubMed Scopus (138) Google Scholar) as well as myomegalin (29Verde I. Pahlke G. Salanova M. Zhang G. Wang S. Coletti D. Onuffer J. Jin S.L. Conti M. J. Biol. Chem. 2001; 276: 11189-11198Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), the p75 neurotrophin receptor (30Sachs B.D. Baillie G.S. McCall J.R. Passino M.A. Schachtrup C. Wallace D.A. Dunlop A.J. MacKenzie K.F. Klussmann E. Lynch M.J. Sikorski S.L. Nuriel T. Tsigelny I. Zhang J. Houslay M.D. Chao M.V. Akassoglou K. J. Cell Biol. 2007; 177: 1119-1132Crossref PubMed Scopus (107) Google Scholar), and SRC family kinases (31McPhee I. Yarwood S.J. Scotland G. Huston E. Beard M.B. Ross A.H. Houslay E.S. Houslay M.D. J. Biol. Chem. 1999; 274: 11796-11810Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Adrenaline exerts many of its actions in regulating cellular signaling processes by increasing the levels of the ubiquitous second messenger, cAMP (32Lefkowitz R.J. Trends Pharmacol. Sci. 2004; 25: 413-422Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 33Lohse M.J. Engelhardt S. Eschenhagen T. Circ. Res. 2003; 93: 896-906Crossref PubMed Scopus (608) Google Scholar). In this a pivotal role is performed by the β2AR, which couples to the G-protein, Gs to activate adenylyl cyclase. This process undergoes rapid desensitization due to the phosphorylation of the β2AR by G-protein receptor kinases, allowing the recruitment of cytosolic β-arrestin to the β2AR, which interdicts its coupling to Gs (34Reiter E. Lefkowitz R.J. Trends Endocrinol. Metab. 2006; 17: 159-165Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 35Gurevich E.V. Gurevich V.V. Genome Biol. 2006; 7: 236Crossref PubMed Scopus (231) Google Scholar). Recently an additional facet of the desensitization process has been uncovered, namely that β-arrestin can sequester the cAMP-specific phosphodiesterase PDE4D5, thereby delivering an active cAMP-hydrolyzing enzyme to the site of cAMP synthesis at the plasma membrane (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (304) Google Scholar). The PDE4D5 isoform preferentially interacts with β-arrestin, with distinct binding sites in both its isoform-specific N-terminal region and its conserved catalytic region (19Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (304) Google Scholar, 23Perry S.J. Baillie G.S. Kohout T.A. McPhee I. Magiera M.M. Ang K.L. Miller W.E. McLean A.J. Conti M. Houslay M.D. Lefkowitz R.J. Science. 2002; 298: 834-836Crossref PubMed Scopus (410) Google Scholar, 24Smith K.J. Baillie G.S. Hyde E.I. Li X. Houslay T.M. McCahill A. Dunlop A.J. Bolger G.B. Klussmann E. Adams D.R. Houslay M.D. Cell. Signal. 2007; 19: 2612-2624Crossref PubMed Scopus (48) Google Scholar, 36Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 37Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 49230-49238Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Thus PDE4D5 straddles β-arrestin, with its unique N-terminal domain interacting with the β-arrestin C-domain and its catalytic unit interacting with the β-arrestin N-domain (38Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (75) Google Scholar). In cardiac myocytes as well as various other primary and transfected cell types, the cytosolic β-arrestin-sequestered PDE4D5 is delivered to plasma membrane localized β2-adrenoceptors (β2ARs) upon agonist challenge (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The sequestered PDE4D5 regulates plasma membrane protein kinase A activity (23Perry S.J. Baillie G.S. Kohout T.A. McPhee I. Magiera M.M. Ang K.L. Miller W.E. McLean A.J. Conti M. Houslay M.D. Lefkowitz R.J. Science. 2002; 298: 834-836Crossref PubMed Scopus (410) Google Scholar), including the phosphorylation of the β2AR by a protein kinase A sub-population tethered to the β2AR by the scaffold, AKAP79 (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 12Willoughby D. Baillie G.S. Lynch M.J. Ciruela A. Houslay M.D. Cooper D.M. J. Biol. Chem. 2007; 282: 34235-34249Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 39Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), conferring a critical and selective regulation upon β2AR signaling (40Bruss M.D. Richter W. Horner K. Jin S.L. Conti M. J. Biol. 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One key β-arrestin partner is Mdm2, which is well known for its critical role in binding p53, thereby sterically blocking the function of its trans-activation domain and acting as an E3 ligase able to ubiquitinate p53 and target it for degradation (46Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Sequestered Mdm2 can also affect β-arrestin ubiquitination (47Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (713) Google Scholar), influencing the stability of β-arrestin interaction with GPCRs, thereby regulating receptor trafficking (48DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1157) Google Scholar, 49Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 14498-14506Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Ubiquitination normally occurs through a three-step process involving Ub-activating (E1), Ub-conjugating (E2), and Ub ligase (E3) enzymes. One means of recognizing transferred ubiquitin moieties on a protein is the presence of the so-called ubiquitin interacting motif (UIM) (50Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). More recently, however, various studies have suggested that a UIM can direct ubiquitination in various proteins and may facilitate mono-ubiquitination (51Miller S.L. Malotky E. O'Bryan J.P. J. Biol. Chem. 2004; 279: 33528-33537Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 52Polo S. Sigismund S. Faretta M. Guidi M. Capua M.R. Bossi G. Chen H. De Camilli P. Di Fiore P.P. Nature. 2002; 416: 451-455Crossref PubMed Scopus (554) Google Scholar, 53Klapisz E. Sorokina I. Lemeer S. Pijnenburg M. Verkleij A.J. van Bergen en Henegouwen P.M. J. Biol. 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This modification is critical for both the internalization of the β2AR and regulation of its levels by lysosomal degradation. Underpinning the ubiquitination of both β-arrestin and G-protein receptor kinase (47Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (713) Google Scholar) is the oncoprotein Mdm2, a well known suppressor of p53 activity (46Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Here we identify, for the first time, the ubiquitination of a cyclic nucleotide phosphodiesterase, namely PDE4D5. Critical for this is a requirement for the β-arrestin-sequestered E3 ligase, Mdm2; a functional UIM located in the C-terminal portion of PDE4D5 plus a priming ubiquitination of PDE4D5 that allows a cascade of ubiquitination within the unique N-terminal region of PDE4D5. This modification enhances the interaction of PDE4D5 with the signaling scaffold protein, β-arrestin, thereby facilitating the fidelity of interaction of β-arrestin with this particular PDE4 isoform. PolyFect transfection reagent and Mdm2 small interference RNA were from Qiagen. Proteases inhibitor mixture tablets were from Roche Applied Science. VSV affinity agarose, anti-VSV antibody, isoprenaline, and N-ethylmaleimide were from Sigma. Mouse monoclonal antibody against ubiquitin (UbP4D1) and anti-Mdm2 antibodies were from Santa Cruz Biotechnology. In vitro ubiquitination kit was from Biomol. Protein G beads were from Amersham Biosciences. ECL was from Pierce. Purified Mdm2 used in vitro ubiquitination assay was a kind gift from Prof. R. Hay (University of Dundee, UK). HEK293 cells stably expressing the V2 receptor were a gift from Dr. R. Lefkowitz (Duke University). PDE4D5 mutants were constructed using QuikChange (Stratagene) and verified by DNA sequencing. HEK293B2 cells are a stable cell line overexpressing the green fluorescent protein-tagged β2AR (58McLean A.J. Milligan G. Br. J. Pharmacol. 2000; 130: 1825-1832Crossref PubMed Scopus (69) Google Scholar), which were cultured and analyzed as described by us previously (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Baillie G.S. Sood A. McPhee I. Gall I. Perry S.J. Lefkowitz R.J. Houslay M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 940-945Crossref PubMed Scopus (304) Google Scholar). HEK293B2 cells were transiently transfected with VSV epitope-tagged forms of one of either wild-type PDE4D5, a β-arrestin binding-defective PDE4D5 (E27A), a RACK-1 binding-defective (L29/30A) PDE4D5, or a UIM-disrupted (E721A:E722A:E723A) PDE4D5. Cell lysis, immunopurification, and Western blotting were done as described before by us in some detail (10Lynch M.J. Baillie G.S. Mohamed A. Li X. Maisonneuve C. Klussmann E. van Heeke G. Houslay M.D. J. Biol. Chem. 2005; 280: 33178-33189Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 36Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 37Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 49230-49238Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). HEK293B2 cells seeded in 100-mm dishes were transfected with the indicated VSV-epitope-tagged PDE4D5 species. After 48 h, cells were challenged with 10 μm isoprenaline for the indicated time. Subsequently, cells were lysed in a 3T3 lysis buffer containing 20 mm HEPES (pH 7.4), 50 mm NaCl, 50 mm NaF, 10% glycerol, 1% Triton X-100, 10 mm EGTA, 30 mm sodium pyrophosphate, 10 mm N-ethylmaleimide, and Roche Applied Science proteases inhibitor mixture. Ubiquitin conjugates were immunopurified on VSV-agarose beads. Ubiquitinated species were detected by immunoblotting using anti-Ubiquitin antibody. PDE4D5 expression levels were determined in cell lysates by Western blotting using specific antibodies. Endogenous ubiquitination analyses were done similarly except that immunopurification was done using a PDE4D5-specific antibody. A Ubiquitination Kit (Biomol) was used, and the procedure was carried out according to the manufacturer's instruction. PDE4D5 peptide array analyses were performed as described previously (36Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 38Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (75) Google Scholar). The membranes were washed with TBST (137 mm NaCl, 20 mm Tris HCl, pH 7.6, 0.1% Tween 20) to stop the ubiquitination reaction. E2-conjugating enzyme UbcH5b and E3 ligase glutathione S-transferase-Mdm2 were used in this assay. The ubiquitin moieties on the peptide array were detected by Western blotting using ubiquitin-specific antibody. This was done as described by us in detail elsewhere (36Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 38Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (75) Google Scholar). Individual PDE4 isoforms are characterized by their unique N-terminal regions. However, although all members of a particular PDE4 sub-family have identical C-terminal regions, these regions show no homology between each of the four different sub-families (5Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (955) Google Scholar, 7Houslay M.D. Adams D.R. Biochem. J. 2003; 370: 1-18Crossref PubMed Scopus (648) Google Scholar). Sequence inspection (Table 1) shows that the sub-family specific C-terminal region of PDE4D contains a potential UIM, for which a 20-residue consensus sequence has been defined of the form: X-Ac-Ac-Ac-Ac-Φ-X-X-Ala-X-X-X-Ser-X-X-Ac-X-X-X-X, where Φ represents a hydrophobic residue, Ac represents an acidic residue, and X represents residues that are less well conserved (59Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). There is no comparable sequence in PDE4 isoforms from the other three sub-families.TABLE 1The unique C-terminal region of PDE4D contains a UIMConsensusXAcAcAcAcΦXXAXXXSXXEXXXXPDE4DvEEEaVge.eeeSqpEacviStam-1kEEEDLakAielSlkEqrqqStam-2kEDEDIakAielSlqEqkqqVps27 (1)dEEElIrkAielSlkEsrnsVps27 (2)eEDpDLkaAiqeSlrEaeeaEps15 (1)sEEDmIewAkreSerEeeqrEps15 (2)qEqEDLelAialSksEiseaHrsqEEEELqlAlalSqsEaeekRap80 (1)tEEEqFalAlkmSeqEarevRap80 (2)eEEElLrkAiaeSlnssggiS5a (1)rqEEEArrAaaaSaaEagiaS5a (2)tEEEgIayAmqmSlggaefgAtaxin3 (1)eDEEDLqrAlalSrqEidmeAtaxin3 (2)dEEaDLrrAiqlSmqgssrnAtaxin3 (3)sEEDmLqaAvtmSletvrndUfo1 (1)nvDEDLqlAialSlsEin..Ufo1 (2)eDDDEFlrAirqSrvEderrUfo1 (3)dEDEqLrrAleeSqliyetqZnf313dEEDmmnqvlqrSiidq...Usp25dDkDDLqrAialSlaEsnraUPL1qEDDELaqAlalSlgnssetKIAA1386eEDpnIllAiqlSlqEsglaRPN10 (1)saDpELalAlrvSmeEqrqrRPN10 (2)tEEEqIayAmqmSlqgaefgMEKK1eEEEaLaiAmamSasqdalpKIAA1386 (1)sEEElLaavleiSkrdaspsKIAA1386 (2)rEEqELqqAlaqSlqEqeawMJD1 (1)eDEEDLqrAlelSrqEidmeMJD1 (2)dEEaDLrrAiqlSmqgssrnMJD1 (3)sEEDmLqaAvtmSletvrnd Open table in a new tab In the PDE4D5 isoform (60Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffmann R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar) this motif is centered upon Val-725, which has a run of three glutamates that are located N-terminal to it (Fig. 1a and Table 1). Positioned six residues C-terminal to Val-725 is located Ser-731, and C-terminal to Ser-731 is Glu-734. This shows good similarity to UIMs identified in various other proteins, save for the lack of an alanine 4 residues N-terminal to Ser-731. Because agonist stimulation of the β2-AR elicits the transient ubiquitination of β-arrestin (47Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (713) Google Scholar) and β-arrestin preferentially sequesters PDE4D5 (37Bolger G.B. McCahill A. Huston E. Cheung Y.F. McSorley T. Baillie G.S. Houslay M.D. J. Biol. Chem. 2003; 278: 4" @default.
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• W2080351377 date "2009-06-01" @default.
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• W2080351377 title "Mdm2 Directs the Ubiquitination of β-Arrestin-sequestered cAMP Phosphodiesterase-4D5" @default.
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• W2080351377 doi "https://doi.org/10.1074/jbc.m109.008078" @default.
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