Mdm2 directs the ubiquitination of beta-arrestin-sequestered cAMP phosphodiesterase-4D5
Authors: George S Baillie
Miles D Houslay
Xiang Li
Journal: Journal of Biological Chemistry (2009)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Beta-arrestin; beta2-adrenoreceptor signaling; adenylyl cyclase; cAMP phosphodiesterase-4D5 (PDE4D5); isoprenaline
Affiliations: Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.

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. 

Retracted: No

PubPeer comments:
  Figure 2 (g) left two lanes are reportedly WT-PDE4D5 Lys (input lysates) and IP (immunoprecipitates). Figure 5 (d) right two lanes are reportedly Triple mutant form under 0 and 10 minute Isoprenaline challenge. The same blot image, one flipped and stretched, used to demonstrate two different experiments. , 
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 The correction is about some text, nothing about the figures. https: www.jbc.org content 284 32 21776.2.short  On page 16170 in the Abstract, the 15th line should read u201cubiquitination of a PDE4D5 spot-immobilized peptide array u2026 u201d , 
 2020 retraction. https: www.jbc.org content 295 34 12328 This article has been withdrawn by the authors. The VSV loading gel immunoblot in Fig. 2G was re-used in Fig. 5D, and the VSV loading gel immunoblot in Fig. 6A was re-used in Fig. S1. The Journal determined that some of the peptide spots in Fig. 7 (C and D) are the same. However, the authors state that similarity is an inevitable occurrence of the robotic u201cspottingu201d system that will produce spots droplets with similar ranges of characteristic shapes over the large panels created. The authors also state that they cannot observe any evidence of u201ccutting and pasting,u201d which they state would be easily observed had it occurred. Notwithstanding these issues, the authors stand by the central tenet of this article, namely that cAMP phosphodiesterase-4D5 (PDE4D5) undergoes multisite ubiquitination. , 
 https: retractionwatch.com 2021 11 11 exclusive-university-of-glasgow-seeking-retraction-of-multiple-papers-after-findings-of-image-manipulation ,