Limits...
Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Uekita T, Itoh Y, Yana I, Ohno H, Seiki M - J. Cell Biol. (2001)

Bottom Line: Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence.MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles.Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Show MeSH

Related in: MedlinePlus

Effect of cytoplasmic mutations on cell surface events mediated by MT1-MMP. (A) CHO-K1 cells were transfected with the expression plasmids for various MT1-MMP mutants. The cells were incubated with purified proMMP-2 in serum-free culture medium. After 18 h, MMP-2 in the culture medium was analyzed by gelatin zymography (top). Cell lysates were subjected to Western blot analysis using the anti–MT1-MMP monoclonal antibody. (B) The cells expressing MT1-F or MT1-F(ΔCP) or mock-transfected cells were incubated with 125I–TIMP-2, and the amount of TIMP-2 bound to the cells was calculated from the radioactivity. (C) Internalization of TIMP-2 bound to the cells was analyzed similarly as described in Fig. 1. Internalization of TIMP-2 bound to the cells expressing MT1-F (▪) or MT1-F(ΔCP) (⋄) is plotted. Values in B and C are the mean ± SD of three experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199326&req=5

fig6: Effect of cytoplasmic mutations on cell surface events mediated by MT1-MMP. (A) CHO-K1 cells were transfected with the expression plasmids for various MT1-MMP mutants. The cells were incubated with purified proMMP-2 in serum-free culture medium. After 18 h, MMP-2 in the culture medium was analyzed by gelatin zymography (top). Cell lysates were subjected to Western blot analysis using the anti–MT1-MMP monoclonal antibody. (B) The cells expressing MT1-F or MT1-F(ΔCP) or mock-transfected cells were incubated with 125I–TIMP-2, and the amount of TIMP-2 bound to the cells was calculated from the radioactivity. (C) Internalization of TIMP-2 bound to the cells was analyzed similarly as described in Fig. 1. Internalization of TIMP-2 bound to the cells expressing MT1-F (▪) or MT1-F(ΔCP) (⋄) is plotted. Values in B and C are the mean ± SD of three experiments.

Mentions: To examine whether MT1-MMP internalization affects MT1-MMP activity on the cell surface, we analyzed the ability of the MT1-MMP mutants to activate proMMP-2, which is one of the important functions of MT1-MMP. MT1-MMP constructs with various mutations in the cytoplasmic domain were expressed in CHO-K1 cells, and their ability to activate the proMMP-2 in the culture medium was examined (Fig. 6 A). None of the mutations altered proMMP-2 activation significantly. Thus, reflecting the expression levels of MT1-MMP mutants on the cell surface (Figs. 3 B and 4 B), their net cell surface proteolytic activity did not change significantly compared with that of the wild-type enzyme.


Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Uekita T, Itoh Y, Yana I, Ohno H, Seiki M - J. Cell Biol. (2001)

Effect of cytoplasmic mutations on cell surface events mediated by MT1-MMP. (A) CHO-K1 cells were transfected with the expression plasmids for various MT1-MMP mutants. The cells were incubated with purified proMMP-2 in serum-free culture medium. After 18 h, MMP-2 in the culture medium was analyzed by gelatin zymography (top). Cell lysates were subjected to Western blot analysis using the anti–MT1-MMP monoclonal antibody. (B) The cells expressing MT1-F or MT1-F(ΔCP) or mock-transfected cells were incubated with 125I–TIMP-2, and the amount of TIMP-2 bound to the cells was calculated from the radioactivity. (C) Internalization of TIMP-2 bound to the cells was analyzed similarly as described in Fig. 1. Internalization of TIMP-2 bound to the cells expressing MT1-F (▪) or MT1-F(ΔCP) (⋄) is plotted. Values in B and C are the mean ± SD of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199326&req=5

fig6: Effect of cytoplasmic mutations on cell surface events mediated by MT1-MMP. (A) CHO-K1 cells were transfected with the expression plasmids for various MT1-MMP mutants. The cells were incubated with purified proMMP-2 in serum-free culture medium. After 18 h, MMP-2 in the culture medium was analyzed by gelatin zymography (top). Cell lysates were subjected to Western blot analysis using the anti–MT1-MMP monoclonal antibody. (B) The cells expressing MT1-F or MT1-F(ΔCP) or mock-transfected cells were incubated with 125I–TIMP-2, and the amount of TIMP-2 bound to the cells was calculated from the radioactivity. (C) Internalization of TIMP-2 bound to the cells was analyzed similarly as described in Fig. 1. Internalization of TIMP-2 bound to the cells expressing MT1-F (▪) or MT1-F(ΔCP) (⋄) is plotted. Values in B and C are the mean ± SD of three experiments.
Mentions: To examine whether MT1-MMP internalization affects MT1-MMP activity on the cell surface, we analyzed the ability of the MT1-MMP mutants to activate proMMP-2, which is one of the important functions of MT1-MMP. MT1-MMP constructs with various mutations in the cytoplasmic domain were expressed in CHO-K1 cells, and their ability to activate the proMMP-2 in the culture medium was examined (Fig. 6 A). None of the mutations altered proMMP-2 activation significantly. Thus, reflecting the expression levels of MT1-MMP mutants on the cell surface (Figs. 3 B and 4 B), their net cell surface proteolytic activity did not change significantly compared with that of the wild-type enzyme.

Bottom Line: Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence.MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles.Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Show MeSH
Related in: MedlinePlus