PT  - JOURNAL ARTICLE
AU  - Da Xu
AU  - Jinghui Zhang
AU  - Qiang Zhang
AU  - Yunzhou Fan
AU  - Chenchang Liu
AU  - Guofeng You
TI  - PKC/Nedd4-2 Signaling Pathway Regulates the Cell Surface Expression of Drug Transporter hOAT1
AID  - 10.1124/dmd.117.075861
DP  - 2017 Jan 01
TA  - Drug Metabolism and Disposition
PG  - dmd.117.075861
4099  - http://dmd.aspetjournals.org/content/early/2017/06/01/dmd.117.075861.short
4100  - http://dmd.aspetjournals.org/content/early/2017/06/01/dmd.117.075861.full
AB  - 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 cell plasma membrane. Short-term activation (<30 min) of protein kinase C (PKC) promotes the attachment of a lysine 48-linked polyubiquitin chain to hOAT1, a process catalyzed by ubiquitin ligase Nedd4-2. The Ubiquitination of hOAT1 then triggers an accelerated endocytosis of the transporter from plasma membrane, which results in a reduced hOAT1 expression at the cell surface and a 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 hrs.) of PKC significantly enhanced hOAT1 degradation, and such action was partially blocked by a ubiquitin mutant Ub-K48R, which has its lysine (K) 48 mutated to arginine (R) and is incapable of forming 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 co-immunoprecipitation 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.