Document Detail

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.
MedLine Citation:
PMID:  11335709     Owner:  NLM     Status:  MEDLINE    
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.
D V Rozanov; E I Deryugina; B I Ratnikov; E Z Monosov; G N Marchenko; J P Quigley; A Y Strongin
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2001-05-02
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  276     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2001 Jul 
Date Detail:
Created Date:  2001-07-09     Completed Date:  2001-08-16     Revised Date:  2013-06-03    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  25705-14     Citation Subset:  IM    
Burnham Institute, La Jolla, California 92037, USA.
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MeSH Terms
Breast Neoplasms / genetics,  metabolism,  pathology
DNA Mutational Analysis
Dipeptides / pharmacology
Enzyme Activation
Fibrosarcoma / genetics,  metabolism,  pathology
Indoles / pharmacology
Matrix Metalloproteinases, Membrane-Associated
Metalloendopeptidases / chemistry,  genetics*,  metabolism
Mutagenesis, Site-Directed
Protease Inhibitors / pharmacology
Protein Processing, Post-Translational
Grant Support
Reg. No./Substance:
0/Dipeptides; 0/Indoles; 0/N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; 0/Protease Inhibitors; 52-90-4/Cysteine; 56-85-9/Glutamine; EC 3.4.24.-/Matrix Metalloproteinases, Membrane-Associated; EC 3.4.24.-/Metalloendopeptidases; I0403ML141/ilomastat

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