Abstract
In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.
Highlights
Intracellular trafficking of MT1-matrix metalloproteinases (MMPs) is essential for its role in tumor cell invasion
These results are consistent with the notion that phosphorylation-dependent internalization and recycling of membrane type 1-matrix metalloproteinase (MT1-MMP) is required for efficient cell migration and invasion, possibly because this trafficking is needed for an association with ␣5 integrin that contributes to activation of ERK
Studies on the endocytosis and trafficking of MT1-MMP reveal the importance of these processes to both cellular invasion and migration [11, 16, 27, 39, 40]
Summary
Intracellular trafficking of MT1-MMP is essential for its role in tumor cell invasion. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Mutation of Thr567 to alanine resulted in increased cell surface expression of MT1-MMP but impaired cellular invasive and migratory abilities. This suggests that the internalization and recycling of MT1-MMP functions to promote invasion and migration through mechanisms beyond ECM degradation. Consistent with this, we found evidence that trafficking/recycling of MT1-MMP is coupled to trafficking of ␣5 integrin and stimulates phosphorylation of ERK
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