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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.

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Internalization-defective MT1-MMP mutants cannot stimulate cell migration. (A) CHO-K1 cells were transfected with the expression plasmids for MT1-F and its mutants. Cell motility was analyzed on colloidal gold–coated coverslips. Representative phagokinetic tracks of the migrating cell are shown after visualization under bright-field illumination. Transfected cells were visualized by immunostaining using anti-FLAG antibody. (B) The migration area of the cell was visualized under bright-field illumination and analyzed using NIH Image software v1.62. The average of 10 cells ± SEM is shown. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant. (C) Internalization of MT1-F during cell migration was monitored. Transfected cells were seeded on coverslips without colloidal gold and incubated. Cell surface MT1-F and its mutant were incubated with Texas red–labeled FLAG M2 antibody. After a 5-min incubation to allow for internalization, surface FLAG M2 antibody was removed by acid wash and internalized FLAG M2 antibody was observed by confocal laser microscopy. Actin was stained with AlexaTM488-conjugated phalloidin. Bars, (A) 100 μm; (B) 10 μm.
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fig8: Internalization-defective MT1-MMP mutants cannot stimulate cell migration. (A) CHO-K1 cells were transfected with the expression plasmids for MT1-F and its mutants. Cell motility was analyzed on colloidal gold–coated coverslips. Representative phagokinetic tracks of the migrating cell are shown after visualization under bright-field illumination. Transfected cells were visualized by immunostaining using anti-FLAG antibody. (B) The migration area of the cell was visualized under bright-field illumination and analyzed using NIH Image software v1.62. The average of 10 cells ± SEM is shown. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant. (C) Internalization of MT1-F during cell migration was monitored. Transfected cells were seeded on coverslips without colloidal gold and incubated. Cell surface MT1-F and its mutant were incubated with Texas red–labeled FLAG M2 antibody. After a 5-min incubation to allow for internalization, surface FLAG M2 antibody was removed by acid wash and internalized FLAG M2 antibody was observed by confocal laser microscopy. Actin was stained with AlexaTM488-conjugated phalloidin. Bars, (A) 100 μm; (B) 10 μm.

Mentions: We observed previously that expression of MT1-MMP promotes the migration of CHO-K1 cells (Kajita et al., 2001). We thus examined whether the internalization-defective mutations also affected the cell migration promoted by MT1-F (Fig. 8). Migration was analyzed by measuring phagokinetic tracks of cells on a coverslip coated with BSA and colloidal gold. Wild-type MT1-F promoted a threefold increase in CHO-K1 cell migration. This phenomenon requires the catalytic activity of MT1-F because the catalytically inactive E/A mutant did not promote the motility at all (Fig. 8, A and B). Interestingly, internalization-defective mutants (ΔCP and L1Y/A) did not stimulate motility, whereas the S/A mutation, which has no effect on internalization, did enhance motility. Other cytoplasmic tail mutants that did not affect internalization also stimulated motility (unpublished data). Thus, the internalization of MT1-F correlates with its ability to promote cell migration. We also confirmed that the internalization defect of MT1-F does not affect the internalization of other cell surface molecules, using hTfnR as a representative molecule (unpublished data).


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)

Internalization-defective MT1-MMP mutants cannot stimulate cell migration. (A) CHO-K1 cells were transfected with the expression plasmids for MT1-F and its mutants. Cell motility was analyzed on colloidal gold–coated coverslips. Representative phagokinetic tracks of the migrating cell are shown after visualization under bright-field illumination. Transfected cells were visualized by immunostaining using anti-FLAG antibody. (B) The migration area of the cell was visualized under bright-field illumination and analyzed using NIH Image software v1.62. The average of 10 cells ± SEM is shown. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant. (C) Internalization of MT1-F during cell migration was monitored. Transfected cells were seeded on coverslips without colloidal gold and incubated. Cell surface MT1-F and its mutant were incubated with Texas red–labeled FLAG M2 antibody. After a 5-min incubation to allow for internalization, surface FLAG M2 antibody was removed by acid wash and internalized FLAG M2 antibody was observed by confocal laser microscopy. Actin was stained with AlexaTM488-conjugated phalloidin. Bars, (A) 100 μm; (B) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Internalization-defective MT1-MMP mutants cannot stimulate cell migration. (A) CHO-K1 cells were transfected with the expression plasmids for MT1-F and its mutants. Cell motility was analyzed on colloidal gold–coated coverslips. Representative phagokinetic tracks of the migrating cell are shown after visualization under bright-field illumination. Transfected cells were visualized by immunostaining using anti-FLAG antibody. (B) The migration area of the cell was visualized under bright-field illumination and analyzed using NIH Image software v1.62. The average of 10 cells ± SEM is shown. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant. (C) Internalization of MT1-F during cell migration was monitored. Transfected cells were seeded on coverslips without colloidal gold and incubated. Cell surface MT1-F and its mutant were incubated with Texas red–labeled FLAG M2 antibody. After a 5-min incubation to allow for internalization, surface FLAG M2 antibody was removed by acid wash and internalized FLAG M2 antibody was observed by confocal laser microscopy. Actin was stained with AlexaTM488-conjugated phalloidin. Bars, (A) 100 μm; (B) 10 μm.
Mentions: We observed previously that expression of MT1-MMP promotes the migration of CHO-K1 cells (Kajita et al., 2001). We thus examined whether the internalization-defective mutations also affected the cell migration promoted by MT1-F (Fig. 8). Migration was analyzed by measuring phagokinetic tracks of cells on a coverslip coated with BSA and colloidal gold. Wild-type MT1-F promoted a threefold increase in CHO-K1 cell migration. This phenomenon requires the catalytic activity of MT1-F because the catalytically inactive E/A mutant did not promote the motility at all (Fig. 8, A and B). Interestingly, internalization-defective mutants (ΔCP and L1Y/A) did not stimulate motility, whereas the S/A mutation, which has no effect on internalization, did enhance motility. Other cytoplasmic tail mutants that did not affect internalization also stimulated motility (unpublished data). Thus, the internalization of MT1-F correlates with its ability to promote cell migration. We also confirmed that the internalization defect of MT1-F does not affect the internalization of other cell surface molecules, using hTfnR as a representative molecule (unpublished data).

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