ABSTRACT
The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here, we examine the interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although S256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at S269. Despite their close proximity, AQP2 phosphorylation at S269 and ubiquitylation at K270 can occur in parallel, with increased S269 phosphorylation and decreased AQP2 endocytosis occurring when K270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occurring in parallel to maximal S269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms might exist for other plasma membrane proteins.
Footnotes
Competing interests
The authors declare no competing interests.
Author contributions
H.B.M.O. developed and designed the study, performed the experiments, analyzed data, wrote the manuscript; T.A. and J.S.H. performed the experiments; T.P. performed the experiments and assisted in the development of the study; R.A.F. developed and designed the study, performed the experiments, analyzed data and wrote the manuscript. All authors approved the final manuscript.
Funding
This work was supported by the Danish Medical Research Council; The Lundbeck Foundation; the Kidney Association (Nyreforeningen); the Novo Nordisk Foundation; the Carlsberg Foundation; and the Aarhus University Forsknings Fond. TP is currently supported by CU Research Cluster: 2014 Ratchadapisek Sompoch Endowment Fund, Chulalongkorn University.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150680/-/DC1
- Received January 26, 2014.
- Accepted May 12, 2014.
- © 2014. Published by The Company of Biologists Ltd
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