Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells

• Published in: The Journal of biological chemistry 2001-07, Vol.276 (28), p.25705
• Main Authors: Rozanov, D V, Deryugina, E I, Ratnikov, B I, Monosov, E Z, Marchenko, G N, Quigley, J P, Strongin, A Y
• Format: Article
• Language: English
• Subjects: Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cysteine; Dimerization; Dipeptides - pharmacology; DNA Mutational Analysis; Enzyme Activation; Female; Fibrosarcoma - genetics; Fibrosarcoma - metabolism; Fibrosarcoma - pathology; Glutamine; Humans; Hydroxamic Acids; Indoles - pharmacology; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Mutagenesis, Site-Directed; Protease Inhibitors - pharmacology; Protein Processing, Post-Translational
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M007921200

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.