The Crumbs complex is an important determinant of epithelial apical-basal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADP-ribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane.
The authors declare no competing or financial interests.
C.I.C. performed or helped perform all experiments, contributed to experimental design and writing of the manuscript. P.S.-T. helped generate samples for BioID, performed the mass spectrometry, and analyzed the results and contributed to writing the manuscript. M.B.-R. helped with the LUMIER assay, provided technical assistance throughout the study and critically read the manuscript. A.D. helped with the LUMIER screen. A.-C.G. contributed to the BioID experiments and critically read the manuscript. J.L.W. designed and supervised the study and helped write the manuscript.
This work was supported by the Canadian Institutes of Health Research Foundation Scheme [grant numbers FDN-143301 to A.C.G., FDN-143252 to J.L.W.], and a Canadian Breast Cancer Foundation (Ontario region) fellowship (to C.I.C.). J.L.W. is a CIBC Chair in Breast Cancer Research.
RAW mass spectrometry data was deposited in the MassIVE repository housed at the Center for Computational Mass Spectrometry at UCSD (http://proteomics.ucsd.edu/ProteoSAFe/datasets.jsp), with the MassIVE ID MSV000079493. It is available for FTP download at: ftp:[email protected] The dataset was assigned the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) identifier PXD003581.
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.188417.supplemental
- Received February 24, 2016.
- Accepted July 22, 2016.
- © 2016. Published by The Company of Biologists Ltd