title: Activation of the cAMP/PKA pathway induces UT-A1 urea transporter monoubiquitination and targets it for lysosomal degradation.
abstract: Regulation of urea transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of the cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkages of the ubiquitin chain. UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis, and internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin-activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK-stimulated UT-A1 degradation occurs through a lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at Ser486 and Ser499 are required for FSK-induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney inner medullary collecting duct suspensions, which showed that vasopressin treatment promotes UT-A1 ubiquitination. We conclude that unlike under basal conditions in which UT-A1 is subject to polyubiquitination and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.
authors.affiliation: Dept. of Physiology, Emory Univ. School of Medicine, Whitehead Research Bldg. Rm. 615, 615 Michael St., Atlanta, GA 30322. gchen3@emory.edu.
authors.forename: Hua
authors.initials: H
authors.lastname: Su
authors.forename: Minguang
authors.initials: M
authors.lastname: Chen
authors.forename: Jeff M
authors.initials: JM
authors.lastname: Sands
authors.forename: Guangping
authors.initials: G
authors.lastname: Chen
mesh_terms: D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005663:Furans / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D026901:Membrane Transport Proteins / Q000378:metabolism*; D023421:Models, Animal; D011720:Pyrazoles / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*; D000096982:Urea Transporters
chemical_list: D001565:Benzoates; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005663:Furans; D026901:Membrane Transport Proteins; D011720:Pyrazoles; D012694:Serine; D000096982:Urea Transporters; C523934:4(4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl)-benzoic acid ethyl ester
keywords: endocytosis; membrane protein; phosphorylation; trafficking; vasopressin