title: Lysine 48-linked polyubiquitination of organic anion transporter-1 is essential for its protein kinase C-regulated endocytosis.
abstract: Organic anion transporter-1 (OAT1) mediates the body's disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We had previously established that OAT1 undergoes constitutive internalization from and recycling back to the cell surface and that acute activation of protein kinase C (PKC) inhibits OAT1 activity by reducing OAT1 cell-surface expression through accelerating its internalization from cell surface to intracellular compartments. However, the underlying mechanisms are poorly understood. In the current study, we provide novel evidence that acute activation of PKC significantly enhances OAT1 ubiquitination both in vitro and ex vivo. We further show that ubiquitination of cell-surface OAT1 increases in cells transfected with dominant negative mutant of dynamin-2, a maneuver blocking OAT1 internalization, which suggests that OAT1 ubiquitination proceeds before OAT1 internalization. Mass spectroscopy has revealed that ubiquitination of OAT1 consists of polyubiquitin chains, primarily through lysine 48 linkage. Transfection of cells with the dominant negative mutant of ubiquitin Ub-K48R, which prevents the formation of Lys48-linked polyubiquitin chains, abolishes PKC-stimulated OAT1 ubiquitination and internalization. Together, our findings demonstrate for the first time that Lys48-linked polyubiquitination is essential for PKC-regulated OAT1 trafficking.
authors.affiliation: Department of Pharmaceutics, Rutgers, the State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Shanshan
authors.initials: S
authors.lastname: Li
authors.forename: Cam
authors.initials: C
authors.lastname: Patterson
authors.forename: Guofeng
authors.initials: G
authors.lastname: You
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D027362:Organic Anion Transport Protein 1 / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D021381:Protein Transport; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D027362:Organic Anion Transport Protein 1; D025801:Ubiquitin; D025821:Polyubiquitin; D011493:Protein Kinase C; D008239:Lysine