Mdm2 directs the ubiquitination of β-arrestin-sequestered cAMP phosphodiesterase-4D5

Xiang Li, George Baillie, Miles Houslay

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

beta-Arrestin plays a key role in regulating beta2-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 beta-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing beta2-adrenoceptors. Reconstitution analyses in beta-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a beta-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 subfamily. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquiti- nation 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 subse- quent cascade of polyubiquitination occurring within its iso- form-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated beta-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 beta-arrestin in cells, thereby contributing to the fidelity of PDE4D5-beta-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.
Original languageEnglish
Pages (from-to)16170-16182
Number of pages13
JournalJournal of Biological Chemistry
Volume284
Issue number24
Early online date16 Apr 2009
DOIs
Publication statusPublished - 12 Jun 2009

Fingerprint

Arrestin
Ubiquitination
Phosphoric Diester Hydrolases
Sumoylation
Mutagenesis
Ubiquitin-Protein Ligases
Fibroblasts
Ubiquitin
RNA Interference
Isoproterenol
Adenylyl Cyclases
Cardiac Myocytes
Adrenergic Receptors

Keywords

  • beta-arrestin
  • phosphodiesterase
  • RACK1
  • ubiquitination
  • Mdm2
  • beta-arrestin-sequestered
  • cAMP Phosphodiesterase-4D5

Cite this

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title = "Mdm2 directs the ubiquitination of β-arrestin-sequestered cAMP phosphodiesterase-4D5",
abstract = "beta-Arrestin plays a key role in regulating beta2-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 beta-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing beta2-adrenoceptors. Reconstitution analyses in beta-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a beta-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 subfamily. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquiti- nation 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 subse- quent cascade of polyubiquitination occurring within its iso- form-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated beta-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 beta-arrestin in cells, thereby contributing to the fidelity of PDE4D5-beta-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.",
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Mdm2 directs the ubiquitination of β-arrestin-sequestered cAMP phosphodiesterase-4D5. / Li, Xiang; Baillie, George; Houslay, Miles.

In: Journal of Biological Chemistry, Vol. 284, No. 24, 12.06.2009, p. 16170-16182.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mdm2 directs the ubiquitination of β-arrestin-sequestered cAMP phosphodiesterase-4D5

AU - Li, Xiang

AU - Baillie, George

AU - Houslay, Miles

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N2 - beta-Arrestin plays a key role in regulating beta2-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 beta-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing beta2-adrenoceptors. Reconstitution analyses in beta-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a beta-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 subfamily. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquiti- nation 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 subse- quent cascade of polyubiquitination occurring within its iso- form-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated beta-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 beta-arrestin in cells, thereby contributing to the fidelity of PDE4D5-beta-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.

AB - beta-Arrestin plays a key role in regulating beta2-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 beta-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing beta2-adrenoceptors. Reconstitution analyses in beta-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a beta-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 subfamily. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquiti- nation 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 subse- quent cascade of polyubiquitination occurring within its iso- form-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated beta-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 beta-arrestin in cells, thereby contributing to the fidelity of PDE4D5-beta-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.

KW - beta-arrestin

KW - phosphodiesterase

KW - RACK1

KW - ubiquitination

KW - Mdm2

KW - beta-arrestin-sequestered

KW - cAMP Phosphodiesterase-4D5

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DO - 10.1074/jbc.M109.008078

M3 - Article

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EP - 16182

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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ER -