Activation of the cAMP/PKA pathway induces UT-A1 urea transporter monoubiquitination and targets it for lysosomal degradation

• Published in: American journal of physiology. Renal physiology Vol. 305; no. 12; pp. F1775 - F1782
• Main Authors: Su, Hua, Chen, Minguang, Sands, Jeff M., Chen, Guangping
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 15.12.2013
• Subjects: Animals; Benzoates - pharmacology; Colforsin - pharmacology; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Dogs; Endocytosis - drug effects; Enzyme Inhibitors - pharmacology; Furans - pharmacology; HEK293 Cells; Humans; Kidney - cytology; Kidney - drug effects; Kidney - metabolism; Kidney diseases; Lysosomes - drug effects; Lysosomes - metabolism; Madin Darby Canine Kidney Cells; Membrane Transport Proteins - metabolism; Models, Animal; Phosphorylation; Plasma; Proteins; Pyrazoles - pharmacology; Rodents; Serine - metabolism; Signal Transduction - physiology; Ubiquitination - drug effects; Ubiquitination - physiology; Urea Transporters; endocytosis; membrane protein; phosphorylation; trafficking; vasopressin
• ISSN: 1931-857X; 1522-1466; 1522-1466
• DOI: 10.1152/ajprenal.00393.2013

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.