TY - JOUR T1 - Three Ubiquitination Sites of Organic Anion Transporter-1 Synergistically Mediate Protein Kinase C-dependent Endocytosis of the Transporter JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.113.086769 SP - mol.113.086769 AU - Shanshan Li AU - Qiang Zhang AU - Guofeng You Y1 - 2013/05/02 UR - http://molpharm.aspetjournals.org/content/early/2013/05/02/mol.113.086769.abstract N2 - Organic anion transporter-1 (OAT1) mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. Therefore, understanding the regulation of OAT1 has profound clinical significance. We previously established that OAT1 constitutively internalizes from and recycles back to cell surface, and that activation of protein kinase C (PKC) inhibits OAT1 activity by promoting ubiquitination of the transporter, which then leads to an accelerated internalization of the transporter from cell surface to intracellular compartments. In the current study, we demonstrated that PKC isoform PKC-alpha was responsible for OAT1 ubiquitination. To directly address the role of OAT1 ubiquitination, we then generated two OAT1 mutants, each having multiple lysines (K) simultaneously mutated to arginine (R). One mutant K163/297/303/315/321R lost sensitivities to PKC-induced inhibition of transport activity, to PKC-induced ubiquitination, and to PKC-induced acceleration of transporter internalization. Further dissecting each lysine within this mutant, we identified Lys297, Lys303 and Lys315 being the ubiquitin-conjugation sites. Interestingly, mutating any one of the three lysines prevented the ubiquitin-conjugation to the other two lysines, suggesting that Lys297, Lys303 and Lys315 may form an optimal structure to interact with ubiquitination machineries. This is the first demonstration that Lys297, Lys303 and Lys315 play synergistic role in PKC-regulated OAT1 ubiquitination, trafficking and transport activity. ER -