title: Biochemical evidence of the interactions of membrane type-1 matrix metalloproteinase (MT1-MMP) with adenine nucleotide translocator (ANT): potential implications linking proteolysis with energy metabolism in cancer cells.
abstract: Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.
authors.affiliation: Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
authors.forename: Ilian A
authors.initials: IA
authors.lastname: Radichev
authors.forename: Albert G
authors.initials: AG
authors.lastname: Remacle
authors.forename: Nor Eddine
authors.initials: NE
authors.lastname: Sounni
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Shiryaev
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Rozanov
authors.forename: Wenhong
authors.initials: W
authors.lastname: Zhu
authors.forename: Natalya V
authors.initials: NV
authors.lastname: Golubkova
authors.forename: Tatiana I
authors.initials: TI
authors.lastname: Postnova
authors.forename: Vladislav S
authors.initials: VS
authors.lastname: Golubkov
authors.forename: Alex Y
authors.initials: AY
authors.lastname: Strongin
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D004734:Energy Metabolism; D005260:Female; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D025941:Protein Interaction Mapping
chemical_list: D000226:Mitochondrial ADP, ATP Translocases; D010447:Peptide Hydrolases; D053511:Matrix Metalloproteinase 14