title: Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1.
abstract: Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey.
authors.forename: Da
authors.initials: D
authors.lastname: Xu
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey.
authors.forename: Haoxun
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey gyou@rci.rutgers.edu.
authors.forename: Guofeng
authors.initials: G
authors.lastname: You
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D027362:Organic Anion Transport Protein 1 / Q000096:biosynthesis* / Q000235:genetics*; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D027362:Organic Anion Transport Protein 1; D034741:RNA, Small Interfering; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C
keywords: Nedd4; drug transport; organic anion transporter; regulation; ubiquitin ligase; ubiquitination
references.citation: Biochem J. 2000 Feb 1;345 Pt 3:503-9
references.pmid: 10642508
references.citation: FASEB J. 2003 Jan;17(1):70-2
references.pmid: 12424229
references.citation: J Biol Chem. 2000 Apr 7;275(14):10278-84
references.pmid: 10744714
references.citation: J Am Soc Nephrol. 2003 Aug;14(8):1959-68
references.pmid: 12874449
references.citation: J Biol Chem. 2004 Feb 6;279(6):5042-6
references.pmid: 14645220
references.citation: Med Res Rev. 2004 Nov;24(6):762-74
references.pmid: 15250040
references.citation: Am J Physiol. 1998 Jan;274(1 Pt 2):F156-64
references.pmid: 9458835
references.citation: Kidney Int. 1998 Jul;54(1):286-300
references.pmid: 9648093
references.citation: Nature. 1999 Aug 12;400(6745):687-93
references.pmid: 10458166
references.citation: Am J Physiol Renal Physiol. 2005 Aug;289(2):F334-46
references.pmid: 15814530
references.citation: Kidney Int. 2005 Dec;68(6):2704-13
references.pmid: 16316345
references.citation: Physiol Rev. 2006 Apr;86(2):669-707
references.pmid: 16601271
references.citation: Arch Biochem Biophys. 2006 Aug 1;452(1):29-37
references.pmid: 16839516
references.citation: J Neurosci. 2006 Aug 2;26(31):8195-205
references.pmid: 16885233
references.citation: J Biol Chem. 2006 Nov 24;281(47):35922-30
references.pmid: 17003038
references.citation: Biochem Pharmacol. 2007 Feb 1;73(3):440-9
references.pmid: 17137557
references.citation: Mol Interv. 2007 Jun;7(3):157-67
references.pmid: 17609522
references.citation: J Biol Chem. 2007 Jul 13;282(28):20207-12
references.pmid: 17502380
references.citation: Am J Physiol Renal Physiol. 2008 May;294(5):F1157-65
references.pmid: 18322022
references.citation: J Biol Chem. 2008 Aug 8;283(32):22166-76
references.pmid: 18544533
references.citation: J Biol Chem. 2008 Nov 21;283(47):32570-9
references.pmid: 18818201
references.citation: Mol Pharmacol. 2009 Sep;76(3):481-90
references.pmid: 19515966
references.citation: Mol Syst Biol. 2009;5:333
references.pmid: 19953087
references.citation: Cell Death Differ. 2010 Jan;17(1):68-77
references.pmid: 19557014
references.citation: J Biol Chem. 2010 Mar 5;285(10):7645-56
references.pmid: 20051513
references.citation: Handb Exp Pharmacol. 2011;(201):29-104
references.pmid: 21103968
references.citation: J Biol Chem. 2010 Dec 3;285(49):38293-303
references.pmid: 20855896
references.citation: J Biol Chem. 2011 Mar 11;286(10):8697-706
references.pmid: 21212261
references.citation: J Biol Chem. 2012 Jun 1;287(23):19177-87
references.pmid: 22505712
references.citation: Mol Pharmacol. 2013 Jan;83(1):217-24
references.pmid: 23087261
references.citation: AAPS J. 2013 Jan;15(1):53-69
references.pmid: 23054972
references.citation: Compr Physiol. 2011 Oct;1(4):1795-835
references.pmid: 23733689
references.citation: Mol Pharmacol. 2013 Jul;84(1):139-46
references.pmid: 23640180
references.citation: Eur J Pharm Sci. 2014 Aug 1;59:94-103
references.pmid: 24747579
references.citation: J Neurosci. 2014 Oct 1;34(40):13371-83
references.pmid: 25274816
references.citation: Arch Pharm Res. 2015;38(5):849-56
references.pmid: 25359200
references.citation: Biochim Biophys Acta. 2015 Oct;1848(10 Pt A):2022-30
references.pmid: 26009271
references.citation: Clin J Am Soc Nephrol. 2015 Nov 6;10(11):2039-49
references.pmid: 26490509
references.citation: Arch Biochem Biophys. 2001 Apr 1;388(1):74-80
references.pmid: 11361144
references.citation: J Biol Chem. 2001 Jul 27;276(30):28321-6
references.pmid: 11359767
references.citation: EMBO J. 2001 Dec 17;20(24):7052-9
references.pmid: 11742982
references.citation: Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25
references.pmid: 11741811
references.citation: Am J Physiol Renal Physiol. 2002 Jul;283(1):F124-33
references.pmid: 12060594
references.citation: Med Res Rev. 2002 Nov;22(6):602-16
references.pmid: 12369090
references.citation: Am J Physiol Renal Physiol. 2002 Nov;283(5):F861-75
references.pmid: 12372761
references.citation: Am J Physiol Renal Physiol. 2000 Jan;278(1):F104-9
references.pmid: 10644661