Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP): The role of the cytoplasmic tail Cys574, the active site Glu 240, and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells

Dmitry V. Rozanov, Elena I. Deryugina, Boris I. Ratnikov, Edward Z. Monosov, George N. Marchenko, James P. Quigley, Alex Y. Strongin

Research output: Contribution to journalArticle

139 Scopus citations

Abstract

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 Cys574 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 tender 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.

Original languageEnglish (US)
Pages (from-to)25705-25714
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
StatePublished - Jul 13 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP): The role of the cytoplasmic tail Cys<sup>574</sup>, the active site Glu <sup>240</sup>, and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells'. Together they form a unique fingerprint.

  • Cite this