Posttranslational regulation of Abcc2 expression by SUMOylation system
Posttranslational regulation of Abcc2 expression by SUMOylation system
复制标题
DOI：
10.1152/ajpgi.90309.2008
复制
发表时间：
2009-02-01
影响因子：
4.5
通讯作者：
Suzuki, Hiroshi
中科院分区：
文献类型：
--
作者：
Minami, Satoko;Ito, Kousei;Suzuki, Hiroshi
Minami S，Ito K，Honma M，Ikebuchi Y，Anzai N，Kanai Y，Nishida T，Tsukita S，Sekine S，Horie T，Suzuki H. SUMO化系统对Abcc2表达的翻译后调控美国生理学杂志胃肠和肝脏生理学296：G406-G413，2009年。首次发表于2008年12月12日; doi：10.1152/ajpgi.90309.2008。ATP结合盒转运蛋白家族C2（Abcc2）是外排转运蛋白家族的一员，参与肝细胞有机阴离子的胆汁排泄。Abcc2的翻译后调节已经被牵连，尽管分子机制还不完全清楚。在本研究中，我们进行了酵母双杂交筛选，以确定新的蛋白质（S），特别是相互作用的接头区之间的NH2-末端核苷酸结合结构域和最后跨膜结构域的Abcc2。筛选导致一系列小泛素样修饰物（SUMO）相关酶及其底物的鉴定。在酵母实验中，通过将Abcc2中的接头区（IKKE）中的推定SUMO共有位点替换为IRKE，所有这些相互作用都被消除。体外SUMO化实验证实，Abcc2接头是Ubc9介导的SUMO化的底物。还发现IKKE序列是SUMO化的靶标，因为具有IKKE的突变体被IRKE取代而不被SUMO化。此外，我们首次证明了Abcc2，内源性表达在大鼠肝癌衍生的McARH7777细胞，SUMO化。内源性Ubc9的小干扰RNA的抑制导致在选择性的30%的减少Abcc2蛋白表达的postnuclear上清液，而亚细胞定位的Abcc2半定量免疫荧光分析证实的影响最小。这是第一次证明通过SUMO化调节ABC转运蛋白表达。
Minami S, Ito K, Honma M, Ikebuchi Y, Anzai N, Kanai Y, Nishida T, Tsukita S, Sekine S, Horie T, Suzuki H. Posttranslational regulation of Abcc2 expression by SUMOylation system. Am J Physiol Gastrointest Liver Physiol 296: G406-G413, 2009. First published December 12, 2008; doi:10.1152/ajpgi.90309.2008.-The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH2-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.