Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

Su, Hua; Carter, Conner B.; Laur, Oskar; Sands, Jeff M.; Chen, Guangping

doi:10.1152/ajprenal.00248.2012

Cited by 12 publications

(18 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell surface protein expression was evaluated using the membrane-impermeable biotinylation reagent NHS-SS-biotin, as previously described [21]. Briefly, cells were seeded on 6-well plates and cultured with 1.2 ml of NHS-SS-biotin (0.6 mg/ml in a borate buffer) in two successive 20-min incubations on ice with very gentle shaking.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose

Lei

Wei

Tang

et al. 2017

Cell Physiol Biochem

Self Cite

View full text Add to dashboard Cite

Background: Protein Kinase C-α (PKC-α) and epidermal growth factor receptor (EGFR) are both involved in diabetic kidney disease; however, the connection between these two proteins during high glucose-induced podocyte injury remains uncertain. Methods: Diabetes was induced in SD rats by streptozotocin (STZ). Fourteen days later, the kidney cortex was removed and subjected to plasma membrane isolation and lipid raft fractionation. In vitro study human podocyte cell line was differentiated and subjected to various treatments. The levels of membranous protein and endocytosis were assessed by biotinylation and sodium 2-mercaptoethane sulfonate (MesNa) treatment. Gö6976 and PYR-41 were used as inhibitors of PKC-α and ubiquitin activating E1 enzyme, respectively. Results: In diabetic rats, the abundance of PKC-α in the membranous fraction and the lipid raft domain is elevated, whereas the EGFR level is reduced. Consistently, in vitro high glucose treated podocytes, membranous EGFR is downregulated with increased PKC-α. Furthermore, the ubiquitination and endocytosis of EGFR are enhanced accompanied by extracellular signal–regulated kinase (ERK) signaling activation and podocyte damage during hyperglycemia. However, these processes can be ameliorated by inhibition of either PKC-α or ubiquitin activating E1 enzyme. Conclusion: During hyperglycemia, PKC-α mediates podocytic EGFR ubiquitination, endocytosis from cell surface and the subsequent ERK activation, which contributes to podocyte injury.

show abstract

Section: Methodsmentioning

confidence: 99%

“…EGFR internalization was measured via biotinylation and sodium 2-mercaptoethane sulfonate (MesNa) treatment [21]. In short, the membranous proteins on the cell surface were first biotin-labeled with a freshly prepared solution of 0.6 mg/ml EZ-Link sulfo-NHS-SS-biotin (Pierce 21331) in a borate buffer as described above.…”

Section: Methodsmentioning

confidence: 99%

PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose

Lei

Wei

Tang

et al. 2017

Cell Physiol Biochem

Self Cite

View full text Add to dashboard Cite

show abstract

“…The pcDNA3 FLAG-Tac-UT-A1, an external FLAGtagged UT-A1, has been described before (29). Amino acids of serine at 486 and 499 of UT-A1 PKA sites were mutated to alanine by site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…We recently found that FSK, the adenylyl cyclase stimulator often being used for in vitro experiments to stimulate UT-A1 urea transport activity, can promote UT-A1 ubiquitination and protein degradation, revealing the coin's other side of UT-A1 phosphorylation by vasopressin or FSK (29). This suggests that activation of the cAMP/PKA pathway causing UT-A1 phosphorylation also triggers the protein ubiquitination and degradation machinery for UT-A1.…”

mentioning

confidence: 99%

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

Chen

Sands

et al. 2013

American Journal of Physiology-Renal Physiology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…We recently reported that FSK, the adenylyl cyclase stimulator often used for in vitro experiments to stimulate UT-A1 urea transport activity, can also promote UT-A1 ubiquitination and protein degradation, revealing the other side of UT-A1 phosphorylation by vasopressin or FSK (29,30). This observation has led to the hypothesis that phosphorylation could be required for UT-A1 ubiquitination.…”

Section: F76mentioning

confidence: 99%

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein

et al. 2015

American Journal of Physiology-Renal Physiology

Self Cite

View full text Add to dashboard Cite

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

show abstract

Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

Cited by 12 publications

References 29 publications

PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose

PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose

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

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein

Contact Info

Product

Resources

About