title: Non-cysteine linked MUC1 cytoplasmic dimers are required for Src recruitment and ICAM-1 binding induced cell invasion.
abstract: BACKGROUND
  The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM-1), which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer cell. Subsequently, we found that MUC1/ICAM-1 binding results in pro-migratory calcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were found to involve Src kinase, a non-receptor tyrosine kinase also implicated in breast cancer initiation and progression. Here, we further investigated the mechanism of MUC1/ICAM-1 signalling, focusing on the role of MUC1 dimerization in Src recruitment and pro-metastatic signalling.
  
  
  METHODS
  To assay MUC1 dimerization, we used a chemical crosslinker which allowed for the detection of dimers on SDS-PAGE. We then generated MUC1 constructs containing an engineered domain which allowed for manipulation of dimerization status through the addition of ligands to the engineered domain. Following manipulation of dimerization, we immunoprecipitated MUC1 to investigate recruitment of Src, or assayed for our previously observed ICAM-1 binding induced events. To investigate the nature of MUC1 dimers, we used both non-reducing SDS-PAGE and generated a mutant construct lacking cysteine residues.
  
  
  RESULTS
  We first demonstrate that the previously observed MUC1/ICAM-1 signalling events are dependent on the activity of Src kinase. We then report that MUC1 forms constitutive cytoplasmic domain dimers which are necessary for Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. The dimers are not covalently linked constitutively or following ICAM-1 binding. In contrast to previously published reports, we found that membrane proximal cysteine residues were not involved in dimerization or ICAM-1 induced signalling.
  
  
  CONCLUSIONS
  Our data implicates non-cysteine linked MUC1 dimerization in cell signalling pathways required for cancer cell migration.
authors.affiliation: Department of Laboratory Medicine and Pathology, 3-70 Heritage Medical Research Centre, University of Alberta, Edmonton, AB, T6G 2S2, Canada.
authors.forename: Ashlyn J
authors.initials: AJ
authors.lastname: Bernier
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Erik
authors.initials: E
authors.lastname: Lillehoj
authors.forename: Andrew R E
authors.initials: AR
authors.lastname: Shaw
authors.forename: Nirosha
authors.initials: N
authors.lastname: Gunasekara
authors.forename: Judith C
authors.initials: JC
authors.lastname: Hugh
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008954:Models, Biological; D018396:Mucin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009361:Neoplasm Invasiveness; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D055503:Protein Multimerization / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018396:Mucin-1; D034741:RNA, Small Interfering; D018799:Intercellular Adhesion Molecule-1; D019061:src-Family Kinases; D003545:Cysteine