Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

BACKGROUND: Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. RESULTS: In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. CONCLUSIONS: Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.