MTCBP-1 binds the CT of MT1-MMP directly to inhibit the invasive properties of pancreatic cancer cells. (A) DanG cells were transfected with GFP or MT1-MMP-GFP, and lysates were immunoprecipitated for MTCBP-1 and blotted for GFP. MT1-MMP-GFP co-immunoprecipitates with MTCBP-1. The asterisk indicates IgG. (B) The purified CT of MT1-MMP (GST-MT1-CT) was incubated with lysate from DanG cells. Endogenous MTCBP-1 co-precipitated with purified GST-MT1-CT, but not GST control. (C) A far-Western blot overlay binding assay was performed to test for a direct interaction between MTCBP-1 and MT1-MMP-CT. MTCBP-1 interacts directly with GST-MT1-CT, but not GST. (D) List of the GST fusion proteins used to map the domains on the CT domain of MT1-MMP that interact with MTCBP-1. (E and F) Far-Western blot overlays were performed to map the domains of direct interaction between MTCBP-1 and the CT domain of MT1-MMP protein (MT1-CT). Depletion of the 10 amino acids at the N terminus (GST-C-10) of MT1-CT abolishes the direct binding between MT1-CT and MTCBP-1. (F) Further analysis using additional truncation forms reveal that residues 568–570 (PRR), are key in mediating this interaction. (G) Functional analysis of the interaction between MT1-MMP and MTCBP-1. Panc-1 cells stably expressing FLAG or MTCBP-1-FLAG were transfected with mCherry control vector, MT1-MMP-mCherry, or MT1-MMP-ΔPRR-mCherry for 48 h and then seeded on green fluorescent gelatin-coated coverslips. Note that overexpression of MTCBP-1 inhibits gelatin degradation induced by MT1-MMP-mCherry, but not MT1-MMP-ΔPRR-mCherry, which cannot bind MTCBP-1. The graphs represent the mean ± SEM of the percentage of cells degrading the matrix (n ≥ 100 cells per condition) or the area of gelatin degradation (n ≥ 10 cells per condition) in three independent experiments. **, P < 0.005; ***, P < 0.0005; ns, not significant. Bars, 20 µm. All blot measurements in kD.