Minami Satoko, Ito Kousei, Honma Masashi, Ikebuchi Yuki, Anzai Naohiko, Kanai Yoshikatsu, Nishida Tamotsu, Tsukita Sachiko, Sekine Shuichi, Horie Toshiharu, Suzuki Hiroshi
Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
Am J Physiol Gastrointest Liver Physiol. 2009 Feb;296(2):G406-13. doi: 10.1152/ajpgi.90309.2008. Epub 2008 Dec 12.
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 NH(2)-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.
ATP结合盒转运蛋白家族C2（Abcc2）是参与肝细胞有机阴离子胆汁排泄的外排转运蛋白成员。虽然Abcc2的翻译后调控已有相关报道，但其分子机制尚未完全明确。在本研究中，我们进行了酵母双杂交筛选，以鉴定与Abcc2位于氨基末端核苷酸结合域和最后一个跨膜域之间的连接区特异性相互作用的新蛋白。筛选结果鉴定出一系列小泛素样修饰物（SUMO）相关酶及其底物。在酵母实验中，通过将Abcc2连接区的假定SUMO共有序列（IKKE）替换为IRKE，所有这些相互作用均被消除。体外SUMO化实验证实，Abcc2连接区是Ubc9介导的SUMO化底物。还发现IKKE序列是SUMO化的靶点，因为IKKE被IRKE替换的突变体未被SUMO化。此外，我们首次证明，在大鼠肝癌来源的McARH7777细胞中内源性表达的Abcc2被SUMO化。用小干扰RNA抑制内源性Ubc9导致核后上清液中Abcc2蛋白表达选择性降低30%，而通过半定量免疫荧光分析证实的Abcc2亚细胞定位受影响最小。这是首次证明SUMO化对ABC转运蛋白表达的调控作用。
