Abstract: SA-PO1062
14-3-3 γ Inhibits BK Activity by Enhancing Its Degradation through a Lysosomal Pathway via an ERK1/2 Signaling-Dependent Mechanism
Session Information
- Na+, K+, Cl-
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Fluid, Electrolytes, and Acid-Base
- 703 Na+, K+, Cl- Basic
Authors
- Chen, Shan, Emory University School of Medicine, Atlanta, Georgia, United States
- Feng, Xiuyan, Emory University School of Medicine, Atlanta, Georgia, United States
- Xiao, Jia, Emory University School of Medicine, Atlanta, Georgia, United States
- Chen, Xinxin, Emory University School of Medicine, Atlanta, Georgia, United States
- Cai, Hui, Emory University School of Medicine, Atlanta, Georgia, United States
Background
14-3-3 γ belongs to a family of multifunction regulatory proteins that mainly bind to phosphorylated Ser/Thr residues in the target proteins. Our previous data showed that 14-3-3 γ inhibits Big K (BK) channel activity and its protein expression through altering ERK1/2 signaling pathway. Thus, we hypothesized that 14-3-3 γ inhibits BK protein expression by altering BK protein degradation via ERK1/2 signaling.
Methods
Cell culture, transfection, western blot analysis, immunoprecipitation, and WT mice were used in this study.
Results
To determine the inhbitory effects of 14-3-3 on BK channel expression, we first did transfection experiments in Cos-7 cells. We found that overexpression of 14-3-3 γ significantly decreased BK protein expression, and knockdown of 14-3-3 γ expression obviously increased BK protein expression. To confirm that 14-3-3 γ modulates BK protein expression through an ERK1/2 signaling pathway, we performed ERK1/2 inhibition experiments. Cos-7 cells were cotransfected with Flag-14-3-3 γ and myc-BK for 48 hours with or without ERK1/2 inhibitior U0126 treatment. We found that inhibition of ERK1/2 phosphorylation abolished 14-3-3 γ-mediated inhibitory effects of BK protein expression. To explore whether overexpression of 14-3-3γ decreased BK protein expression through a lysosomal degradation pathway, we determined the effects of lysosomal inhibitor, bafilomycin A1 (Baf A1) on BK protein expression in Cos-7 cells. We found that Baf A1 treatments reversed the inhibitory effects of 14-3-3 γ on BK protein expression. We further investigated whether 14-3-3 γ involved in BK protein ubiquitination in HEK 293 stably expressing BK cells transiently transfected with 14-3-3 γ plasmid. We found that overexpression of 14-3-3 γ increased BK protein ubiquitination while increasing ERK 1/2 phosphorylation.
Conclusion
These data suggested that 14-3-3 γ inhibits BK protein expression by increasing BK ubiquitination, leading to enhanced BK degradation through a lysosomal pathway via an ERK1/2 signaling-dependent mechanism.
Funding
- Veterans Affairs Support