Figure 1.

Expression of MT1-MMP cytoplasmic tail mutants in ovarian cancer cells. A, schematic illustration of wild-type and cytoplasmic tail mutations of MT1-MMP. Structural domains of MT1-MMP are shown from the N terminus: signal peptide (SP), pro-peptide that contains a basic RRKR motif cleaved by furin (Pro), catalytic domain that contains the zinc-binding site (Zn²⁺) (CAT), hinge region (H), hemopexin-like domain (HPX), linker (L), transmembrane domain (TM), and cytoplasmic tail domain (20 amino acids) (CT). Thr⁵⁶⁷ in the cytoplasmic tail was mutated either to Glu to make a phosphomimetic mutant (TE) or to Ala to generate a phosphodefective mutant (TA) (22, 23). B, cell lines were generated in an OVCA433 parental background expressing GFP-tagged MT, TE, and TA. Western blotting was performed to detect MT1-MMP, E-cadherin (E-cad), and β-actin as a loading control. Cell lysates (20 μg) were electrophoresed on 9% SDS-polyacrylamide gels and electroblotted to Immobilon as described under “Experimental procedures.” Blots were probed with anti-MT1-MMP (1:2,000 dilution) or anti-E-cadherin (1:5,000 dilution) as indicated and developed using a peroxidase-conjugated secondary antibody (1:4,000 dilution) and ECL detection, as described under “Experimental procedures.” Blots were stripped and reprobed with anti-β-actin-peroxidase antibody (1:100,000 dilution) to ensure equal loading. Top, arrow, 55-kDa active MT1-MMP; arrowhead, 82-kDa GFP-tagged MT1-MMP. Second panel, arrow, 120-kDa intact E-cadherin; arrowhead, 100-kDa cleaved E-cadherin. C, densitometric analysis of cleaved E-cadherin. Experiments were repeated in biologic triplicates. **, p < 0.01.