Mdm2 Directs the Ubiquitination of β-Arrestin-sequestered cAMP Phosphodiesterase-4D5 (Retracted Article)
Mdm2 Directs the Ubiquitination of β-Arrestin-sequestered cAMP Phosphodiesterase-4D5 (Retracted Article)
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DOI：
10.1074/jbc.m109.008078
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发表时间：
2009-06-12
影响因子：
4.8
通讯作者：
Houslay, Miles D.
中科院分区：
文献类型：
--
作者：
Li, Xiang;Baillie, George S.;Houslay, Miles D.
β -阻滞素在调节β(2)-肾上腺素受体信号传导中发挥关键作用，通过阻断腺苷酸环化酶的激活和选择性隔离cAMP磷酸二酯酶- 4d5 (PDE4D5)，将活性cAMP降解系统传递到cAMP合成位点。在这里，我们发现β受体激动剂异丙肾上腺素在原代心肌细胞、小鼠胚胎成纤维细胞和组成性表达β(2)-肾上腺素受体的HEK293B2细胞中触发PDE4D5的快速和短暂泛素化。对β -arrestin1/2双敲除细胞的重构分析和小干扰RNA敲除研究表明，β -arrestin1 - 2支架池的e3泛素连接酶Mdm2介导PDE4D5泛素化。关键是位于PDE4D5末端C端的泛素相互作用基序，这是PDE4D亚家族所特有的。对PDE4D5点固定化肽阵列进行体外SUMOylation，然后进行诱变策略，结果表明PDE4D5泛素化发生在位于其亚型特异性n端区域的Lys-48、Lys- 53和Lys-78以及位于其调控模块UCR1的Lys-140上。我们认为，Lys-140的单泛素化启动了PDE4D5，随后在Lys-48、Lys-53和Lys-78的同工异构体特异性n端区域发生了级联的多泛素化。PDE4D5与非泛素化β -抑制蛋白亚群相互作用，由于隔离的PDE4D5引起的空间位阻，该亚群可能受到mdm2介导的泛素化的保护。PDE4D5的泛素化引起细胞中被β -抑制素隔离的PDE4D5的比例增加，从而有助于PDE4D5- β -抑制素相互作用的保真度，并降低被支架蛋白RACK1隔离的PDE4D5的比例。
beta-Arrestin plays a key role in regulating beta(2)-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 beta(2)-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 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 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.