Three ubiquitination sites of organic anion transporter-1 synergistically mediate protein kinase C-dependent endocytosis of the transporter.

Organic anion transporter-1 (OAT1) mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, 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 showed that PKC isoform PKCα 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 in this mutant, we identified Lys297, Lys303, and Lys315 as being the ubiquitin conjugation sites. Of interest, 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 a synergistic role in PKC-regulated OAT1 ubiquitination, trafficking, and transport activity.