Mdm2 directs the ubiquitination of beta-arrestin-sequestered cAMP phosphodiesterase-4D5.
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 sub-family. In vitro ubiquitination [corrected] 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 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.
Li
Xiang
X
From Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Baillie
George S
GS
From Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Houslay
Miles D
MD
From Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom. Electronic address: M.Houslay@bio.gla.ac.uk.
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002478:Cells, Cultured; D016384:Consensus Sequence; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D005347:Fibroblasts / Q000166:cytology; D019204:GTP-Binding Proteins / Q000378:metabolism; D055786:Gene Knockout Techniques; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008239:Lysine / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology; D009363:Neoplasm Proteins / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects; D014667:Vasopressins / Q000494:pharmacology; D000071557:beta-Arrestins
D000318:Adrenergic beta-Agonists; D019390:Arrestins; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D025801:Ubiquitin; D000071557:beta-Arrestins; D014667:Vasopressins; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D010727:Phosphoric Diester Hydrolases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D019204:GTP-Binding Proteins; D008239:Lysine; D007545:Isoproterenol