Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells
1 Molecular Oncology Laboratory, Oncology Research Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, 2145 NSW, Australia
2 The University of Sydney Discipline of Paediatrics and Child Health, The Children's Hospital at Westmead, Locked Bag 4001, Westmead 2145, NSW, Australia
3 Department of Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
4 Epithelial Cancer and Mucosal Biology Laboratory, Mater Medical Research Institute, Mater Health Services, South Brisbane 4101 Qld, Australia
5 Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049-Madrid, Spain
Citation and License
BMC Cell Biology 2009, 10:7 doi:10.1186/1471-2121-10-7Published: 28 January 2009
The MAL2 gene, encoding a four-transmembrane protein of the MAL family, is amplified and overexpressed in breast and other cancers, yet the significance of this is unknown. MAL-like proteins have trafficking functions, but their molecular roles are largely obscure, partly due to a lack of known binding partners.
Yeast two-hybrid screening of a breast carcinoma cDNA expression library was performed using a full-length MAL2 bait, and subsequent deletion mapping experiments were performed. MAL2 interactions were confirmed by co-immunoprecipitation analyses and confocal microscopy was employed to compare protein sub-cellular distributions. Sucrose density gradient centrifugation of membranes extracted in cold Triton X-100 was employed to compare protein distributions between Triton X-100-soluble and -insoluble fractions.
The tumor-associated protein mucin 1 (MUC1) was identified as a potential MAL2 partner, with MAL2/MUC1 interactions being confirmed in myc-tagged MAL2-expressing MCF-10A cells using co-immunoprecipitation assays. Deletion mapping experiments demonstrated a requirement for the first MAL2 transmembrane domain for MUC1 binding, whereas the MAL2 N-terminal domain was required to bind D52-like proteins. Confocal microscopy identified cytoplasmic co-localisation of MUC1 and MAL2 in breast cell lines, and centrifugation of cell lysates to equilibrium in sucrose density gradients demonstrated that MAL2 and MUC1 proteins were co-distributed between Triton X-100-soluble and -insoluble fractions. However co-immunoprecipitation analyses detected MAL2/MUC1 interactions in Triton X-100-soluble fractions only. Myc-MAL2 expression in MCF-10A cells was associated with both increased MUC1 detection within Triton X-100-soluble and -insoluble fractions, and increased MUC1 detection at the cell surface.
These results identify MUC1 as a novel MAL2 partner, and suggest a role for MAL2 in regulating MUC1 expression and/or localisation.