PKC/Nedd4-2 Signaling Pathway Regulates the Cell Surface Expression of Drug Transporter hOAT1

Human organic anion transporter-1 (hOAT1) regulates the absorption, distribution, and excretion of a wide range of clinically important drugs. Our previous work demonstrated that hOAT1 is a dynamic membrane transporter, constitutively internalizing from and recycling back to the cell plasma membrane. Short-term activation (<30 minutes) of protein kinase C (PKC) promotes the attachment of a lysine 48-linked polyubiquitin chain to hOAT1, a process catalyzed by ubiquitin ligase neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2). The ubiquitination of hOAT1 then triggers an accelerated endocytosis of the transporter from plasma membrane, which results in reduced hOAT1 expression at the cell surface and decreased hOAT1 transport activity. In the present study, we investigated the long-term effect of PKC on hOAT1. We showed that long-term activation (>2 hours) of PKC significantly enhanced hOAT1 degradation, and such action was partially blocked by ubiquitin mutant Ub-K48R, which has its lysine (K) 48 mutated to arginine (R) and is incapable of forming a K48-linked polyubiquitin chain. The ubiquitin ligase Nedd4-2 was also found to augment hOAT1 degradation. These results suggest that PKC-regulated and Nedd4-2-catalyzed attachment of a lysine 48-linked polyubiquitin chain to hOAT1 is important for hOAT1 stability. We further showed through coimmunoprecipitation experiments that there was a direct association between hOAT1 and Nedd4-2, and such interaction was weakened when the WW3 and WW4 domains of the ligase were mutated. Mutating WW3 and WW4 domains of the ligase also impaired its ability to ubiquitinate hOAT1. Therefore, WW3 and WW4 domains of Nedd4-2 are critical for its association with and modulation of the transporter.