Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
These results identify MUC1 as a novel MAL2 partner, and suggest a role for MAL2 in regulating MUC1 expression and/or localisation.
Fanayan
Susan
S
Molecular Oncology Laboratory, Oncology Research Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, 2145 NSW, Australia. sfanayan@cbms.mq.edu.au
Shehata
Mona
M
Agterof
Annelies P
AP
McGuckin
Michael A
MA
Alonso
Miguel A
MA
Byrne
Jennifer A
JA
D001943:Breast Neoplasms / Q000378:metabolism*; D002277:Carcinoma / Q000378:metabolism*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D018396:Mucin-1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D011510:Proteolipids / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; C438606:MAL2 protein, human; D018396:Mucin-1; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D011510:Proteolipids; D033921:Vesicular Transport Proteins