title: Three ubiquitination sites of organic anion transporter-1 synergistically mediate protein kinase C-dependent endocytosis of the transporter.
abstract: 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.
authors: Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Rd., Piscataway, NJ 08854, USA.
authors: Shanshan
authors: S
authors: Li
authors: Qiang
authors: Q
authors: Zhang
authors: Guofeng
authors: G
authors: You
mesh_terms: D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004705:Endocytosis; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics; D027362:Organic Anion Transport Protein 1 / Q000235:genetics* / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D027362:Organic Anion Transport Protein 1; D025801:Ubiquitin; D001120:Arginine; D011493:Protein Kinase C; D008239:Lysine
references: J Biol Chem. 2005 Oct 21;280(42):35617-24
references: 16109712
references: Kidney Int. 1998 Jul;54(1):286-300
references: 9648093
references: Biochim Biophys Acta. 2003 Dec 30;1618(2):185-93
references: 14729155
references: Med Res Rev. 2002 Nov;22(6):602-16
references: 12369090
references: J Biol Chem. 2007 Jul 13;282(28):20207-12
references: 17502380
references: Kidney Int. 2005 Dec;68(6):2704-13
references: 16316345
references: Am J Physiol Renal Physiol. 2000 Jan;278(1):F104-9
references: 10644661
references: J Biol Chem. 2009 Jul 10;284(28):18778-89
references: 19398555
references: Biopharm Drug Dispos. 2010 Jan;31(1):1-71
references: 19953504
references: Am J Physiol. 1998 Jan;274(1):F156-64
references: 9458835
references: Mol Pharmacol. 2013 Jan;83(1):217-24
references: 23087261
references: Am J Physiol Renal Physiol. 2002 Nov;283(5):F861-75
references: 12372761
references: Am J Physiol Renal Physiol. 2002 Jul;283(1):F124-33
references: 12060594
references: Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25
references: 11741811
references: Mol Pharmacol. 2009 Sep;76(3):481-90
references: 19515966
references: Xenobiotica. 2008 Jul;38(7-8):889-935
references: 18668434
references: Arch Biochem Biophys. 2001 Apr 1;388(1):74-80
references: 11361144
references: J Biol Chem. 2008 Nov 21;283(47):32570-9
references: 18818201
references: Biochemistry. 2001 May 8;40(18):5511-20
references: 11331016
references: Arch Biochem Biophys. 2006 Aug 1;452(1):29-37
references: 16839516
references: Mol Cell Biol. 2008 Sep;28(17):5275-87
references: 18573876
references: J Cell Biol. 2007 Dec 3;179(5):935-50
references: 18056411
references: Cell Biochem Biophys. 2002;36(2-3):169-74
references: 12139402
references: J Biol Chem. 2004 Apr 9;279(15):14961-6
references: 14749323
references: Am J Physiol Endocrinol Metab. 2009 Feb;296(2):E378-83
references: 19088254