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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 9, 2005 as doi:10.1096/fj.04-3651fje. |
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,1
* Department of Matrix Biology, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London, UK;
Division of Cancer Cell Research, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan; and
Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, UK
2Correspondence: Department of Matrix Biology, Kennedy Institute of Rheumatology Division, Imperial College London, 1 Aspenlea Road, London W6 8LH, UK. E-mail: y.itoh{at}imperial.ac.uk
SPECIFIC AIMS
MT1-MMP is a type I transmembrane proteinase that promotes cell migration and invasion. The enzyme is regulated by different means including inhibitors, homophilic complex formation, localization to migration front, and internalization from the cell surface. The aim of this study was to test whether MT1-MMP is post-translationally modified by palmitate and to investigate the role of this lipid modification in the biological function of the enzyme.
PRINCIPAL FINDINGS
The importance of the cytoplasmic domain of MT1-MMP for its internalization and cell migration-promoting activity is well documented. In the cytoplasmic domain, there is a single cystein at position 574. This cysteine has been described to form a disulfide bond with a Cys574 of a neighboring MT1-MMP. On the other hand, such a cysteine at a juxtamembranous region is also a candidate for palmitoylation. We found that 1) Cys574 of MT1-MMP is not forming a disulfide bond to form a dimer but is palmitoylated, 2) palmitoylation is essential for its cell migration-promoting activity and its clathrin-dependent internalization, and 3) the position of palmitoylated cysteine is critical for clathrin-mediated internalization and MT1-MMP-mediated cell motility.
1. MT1-MMP is palmitoylated at Cys574
To test whether MT1-MMP can form an SDS-resistant dimer through Cys574 of neighboring MT1-MMPs, Cys574 was substituted to either alanine or serine (Fig. 1
A). Analysis of these mutants together with the wild-type enzyme expressed in COS7 cells indicated there are no SDS-resistant Cys574-mediated dimers (Fig. 1B
). Endogenous MT1-MMP expressed in HT1080 cells also does not form SDS-resistant dimers (Fig. 1B
). Therefore, we concluded that MT1-MMP does not form a disulfide bond-mediated dimer. Next we tested whether Cys574 is palmitoylated by metabolic labeling experiments with [3H]palmitate in COS7 cells or HT1080 cells (Fig. 1C
). The data demonstrate that wild-type MT1-MMP, but not C574A or C574S mutant, is palmitoylated in both cell lines. These data show that MT1-MMP is palmitoylated at Cys574.
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2. A role of palmitoylation for the function of MT1-MMP
We next investigated the role of palmitoylation for the function of MT1-MMP. Palmitoylation is not essential for proMMP-2 activation as both C574A and C574S mutants activate proMMP-2 as well as wild-type. However, expression of the C574A mutant or the cytoplasmic domain deletion mutants that lack Cys574 failed to promote cell motility whereas wild-type did. The ability of the enzymes to promote cell motility coincides with rapid internalization through a clathrin-mediated pathway. C574A and deletion mutants of MT1-MMP that failed to promote cell motility were internalized much slower than the wild-type, and this internalization was found to be through the caveolae-mediated pathway. The data suggest that palmitoylation of MT1-MMP is important for its clathrin-mediated internalization and that clathrin-mediated internalization, but not caveolae-mediated internalization, is essential to promote cell motility.
3. The position of palmitoylated cysteine relative to the LLY573 sequence is critical for clathrin-mediated MT1-MMP internalization and MT1-MMP-mediated cell migration
To study whether the position of the palmitoylated cysteine within the cytoplasmic tail is important, a cysteine was inserted at different positions of the cytoplasmic tail of the C574A mutant of MT1-MMP (Fig. 2
A). We have previously shown that LLY573 is a motif that interacts with the µ2 subunit of adaptor protein 2, which mediates the incorporation of MT1-MMP into the clathrin cages for internalization. The result indicates that the position of the cysteine relative to transmembrane domain is important for palmitoylation as a cysteine positioned 18 amino acids away from the transmembrane domain (580C) was partially palmitoylated, whereas a cysteine 21 amino acid away was not palmitoylated at all (Fig. 2B
). These mutants were analyzed for their internalization (Fig. 2C
) and cell migration-promoting activity (Fig. 2D
). As the partially palmitoylated 580C mutant was not internalized well and did not promote cell motility, it became clear that the position of the palmitoylated cysteine relative to LLY573 may be important for MT1-MMP’s internalization and cell motility-promoting activity.
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CONCLUSIONS AND SIGNIFICANCE
We report here that MT1-MMP is post-translationally modified by C16 palmitate at Cys574 in its cytoplasmic domain. The palmitoylation is essential for MT1-MMP’s cell motility-promoting activity and the internalization through a clathrin-mediated pathway. We previously reported that the LLY573 motif, which interacts with the µ2 subunit of adaptor protein 2 (AP2), facilitates incorporation of the enzyme into clathrin-coated pits. Because Cys574 is located immediately C-terminal to this motif, attachment of a lipid that integrates into the plasma membrane positions the LLY573 motif immediately proximal to the plasma membrane. Our mutation results also indicated that palmitoylated cysteine has to be positioned close to the LLY573 motif for the enzyme to be internalized via the clathrin pathway. It is possible that the membrane proximity of LLY573 determines the efficacy of the interaction of the LLY573 motif with the µ2 subunit of AP-2 (Fig. 3
). Another possibility is that palmitoylation does not affect the interaction of µ2 with LLY573, but is the resulting membrane proximity that is essential for the MT1-MMP-AP-2 complex to be effectively incorporated into a clathrin cage. In either case, secondary or tertiary structure induced by palmitoylation is the key.
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Palmitoylation is a reversible lipid modification of proteins, and transient palmitoylation has been shown to play a role in signal transduction pathways such as G-protein-coupled receptor signaling. It has been suggested that all S-acylated proteins are likely to be subjected to a thioacylation-deacylation cycle. This suggested there may be palmitoylated and de-palmitoylated MT1-MMP on the cell surface and that the palmitoylation step could be a regulatory mechanism of the function of MT1-MMP (Fig. 3)
. Since palmitoylation-deficient mutants were neither internalized through the clathrin pathway nor promoted cell migration, depalmitoylation of MT1-MMP would down-regulate its function relating to cell migration without modifying the proteolytic activity of the enzyme. Thus, elucidation of the MT1-MMP’s thioacylation regulatory cycle may assist us to further understand the role of MT1-MMP in cell motility.
FOOTNOTES
1 Present address: Growth Factor Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3651fje;
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