PMID:19372219

Citation	Li, X, Baillie, GS and Houslay, MD (2009) Mdm2 directs the ubiquitination of beta-arrestin-sequestered cAMP phosphodiesterase-4D5. J. Biol. Chem. 284:16170-82
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 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.
Links	PubMed PMC2713507 Online version:10.1074/jbc.M109.008078
Keywords	Adrenergic beta-Agonists/pharmacology; Animals; Arrestins/metabolism; Cells, Cultured; Consensus Sequence; Cyclic Nucleotide Phosphodiesterases, Type 3; Fibroblasts/cytology; GTP-Binding Proteins/metabolism; Gene Knockout Techniques; Humans; Isoproterenol/pharmacology; Kidney/cytology; Lysine/metabolism; Mice; Myocytes, Cardiac/cytology; Neoplasm Proteins/metabolism; Phosphoric Diester Hydrolases/chemistry; Phosphoric Diester Hydrolases/genetics; Phosphoric Diester Hydrolases/metabolism; Protein Structure, Tertiary; Proto-Oncogene Proteins c-mdm2/genetics; Proto-Oncogene Proteins c-mdm2/metabolism; Rats; Receptors, Cell Surface/metabolism; Transfection; Ubiquitin/metabolism; Ubiquitination/drug effects; Vasopressins/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MOUSE:MDM2	enables	GO:0097110: scaffold protein binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:P32121	F		Seeded From UniProt	complete
HUMAN:MDM2	enables	GO:0061630: ubiquitin protein ligase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F	has_direct_input:(UniProtKB:Q08499-6)	Seeded From UniProt	complete
HUMAN:MDM2	involved_in	GO:0018205: peptidyl-lysine modification	ECO:0000315: mutant phenotype evidence used in manual assertion		P	has_direct_input:(UniProtKB:Q08499-6)	Seeded From UniProt	complete
HUMAN:ARRB2	involved_in	GO:0031398: positive regulation of protein ubiquitination	ECO:0000316: genetic interaction evidence used in manual assertion	UniProtKB:Q91YI4	P		Seeded From UniProt	complete
HUMAN:ARRB2	enables	GO:0031625: ubiquitin protein ligase binding	ECO:0000353: physical interaction evidence used in manual assertion	UniProtKB:P23804	F		Seeded From UniProt	complete
HUMAN:ARRB2	involved_in	GO:0060765: regulation of androgen receptor signaling pathway	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:ARRB2	enables	GO:0060090: molecular adaptor activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:V2R	involved_in	GO:0031398: positive regulation of protein ubiquitination	ECO:0000303: author statement without traceable support used in manual assertion		P		Seeded From UniProt	complete

References

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