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Forskolin stimulation promotes urea transporter UT‐A1 ubiquitination, endocytosis and degradation in MDCK cells
Author(s) -
Chen Guangping,
Su Hua,
Carter Conner B,
Fröhlich Otto,
Sands Jeff M
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1067.5
Subject(s) - forskolin , microbiology and biotechnology , cycloheximide , brefeldin a , endocytosis , ubiquitin ligase , mg132 , chemistry , protein degradation , proteasome inhibitor , biology , proteasome , stimulation , biochemistry , ubiquitin , cell , endocrinology , golgi apparatus , protein biosynthesis , endoplasmic reticulum , gene
Adenylyl cyclase stimulator forskolin (FSK) stimulates UT‐A1 phosphorylation, membrane trafficking as well as urea transport activity. Here, we found that forskolin stimulation induces UT‐A1 ubiquitination in UT‐A1 MDCK cells. This suggests that phosphorylation by FSK also activates the UT‐A1 protein degradation machinery. UT‐A1 MDCK cells were treated with 100μg/ ml cycloheximide to inhibit proteins synthesis, with or without 10 μM FSK. Total UT‐A1 protein abundance was significantly reduced after FSK treatment. We then specifically examined the degradation of cell surface UT‐A1 by cell surface biotinylation. UT‐A1 MDCK cells were pretreated with 2 μM brefeldin A (BFA) for 2 h to block the newly produced protein from trafficking to the cell membrane. FSK treatment accelerated UT‐A1 removal from the cell plasma membrane. Proteasome inhibitor MG132 reduced FSK‐induced membrane UT‐A1 reduction. The reduced cell membrane UT‐A1 is due to an increase in UT‐A1 endocytosis as judged by biotinylation and MesNa treatment. We further found that UT‐A1 activation/phosphorylation by FSK promotes its binding to 14–3‐3γ and ubiquitin E3 ligase MDM2. Our study shows that FSK activates UT‐A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation mechanism of UT‐A1 which is important for the cells to limit the reaction in response to stimulation and enable the cells back to basal conditions.