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.
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.
Rozanov
D V
DV
Burnham Institute, La Jolla, California 92037, USA.
Deryugina
E I
EI
Ratnikov
B I
BI
Monosov
E Z
EZ
Marchenko
G N
GN
Quigley
J P
JP
Strongin
A Y
AY
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D003545:Cysteine; D004252:DNA Mutational Analysis; D019281:Dimerization; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D005354:Fibrosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005973:Glutamine; D006801:Humans; D006877:Hydroxamic Acids; D007211:Indoles / Q000494:pharmacology; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational
D004151:Dipeptides; D006877:Hydroxamic Acids; D007211:Indoles; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D005973:Glutamine; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C119203:ilomastat; D003545:Cysteine