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LIVER AND BILIARY TRACT

Posttranslational regulation of Abcc2 expression by SUMOylation system

¹Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo; ²Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo; ³Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka; ⁴Department of Human Functional Genomics, Life Science Research Center, Mie University, Tsu, Mie; ⁵Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Suita, Osaka; and ⁶Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba, Japan

Submitted 28 April 2008 ; accepted in final form 10 December 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.

MRP2; ABC transporter; Ubc9