Membrane-type 1 Matrix Metalloproteinase Cytoplasmic Tail-binding Protein-1 Is a New Member of the Cupin Superfamily

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is an enzyme that promotes tumor cell invasion in tissues. Although the proteolytic activity of MT1-MMP is indispensable for invasion, it is also regulated by functions of the cytoplasmic tail. In this study we obtained a new human gene whose product binds to the tail sequence in yeast. The product, MTCBP-1, is a 19-kDa protein that belongs to the newly proposed Cupin superfamily composed of proteins with diverse functions. MTCBP-1 expressed in cells formed a complex with MT1-MMP and co-localized at the membrane. It was also detected in both the cytoplasm and nucleus, where MT1-MMP does not exist. In human tumor cell lines MTCBP-1 expression was significantly low compared with non-transformed fibroblasts, and enforced expression of MTCBP-1 inhibited the activity of MT1-MMP in promoting cell migration and invasion. MTCBP-1 showed significant homology to the bacterial aci-reductone dioxygenase, which is an enzyme in methionine metabolism. The C-terminal part of MTCBP-1 is identical to Sip-L, which is reported to be important for human hepatitis C virus replication. Thus, MTCBP-1 may have multiple functions other than the regulation of MT1-MMP, which presumably depends on the subcellular compartment. Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is an enzyme that promotes tumor cell invasion in tissues. Although the proteolytic activity of MT1-MMP is indispensable for invasion, it is also regulated by functions of the cytoplasmic tail. In this study we obtained a new human gene whose product binds to the tail sequence in yeast. The product, MTCBP-1, is a 19-kDa protein that belongs to the newly proposed Cupin superfamily composed of proteins with diverse functions. MTCBP-1 expressed in cells formed a complex with MT1-MMP and co-localized at the membrane. It was also detected in both the cytoplasm and nucleus, where MT1-MMP does not exist. In human tumor cell lines MTCBP-1 expression was significantly low compared with non-transformed fibroblasts, and enforced expression of MTCBP-1 inhibited the activity of MT1-MMP in promoting cell migration and invasion. MTCBP-1 showed significant homology to the bacterial aci-reductone dioxygenase, which is an enzyme in methionine metabolism. The C-terminal part of MTCBP-1 is identical to Sip-L, which is reported to be important for human hepatitis C virus replication. Thus, MTCBP-1 may have multiple functions other than the regulation of MT1-MMP, which presumably depends on the subcellular compartment. Membrane-type 1 matrix metalloproteinase (MT1-MMP) 1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; RT, reverse transcription; TIMP, tissue inhibitor of metalloproteinase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; HPX domain, hemopexin-like domain; MTCBP-1, MT1-MMP cytoplasmic tail-binding protein-1; rMTCBP-1, recombinant MTCBP-1; ARD, aci-reductone dioxygenase; DSBH, double-stranded β helix; kb, kilobases; WT, wild type. 1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; RT, reverse transcription; TIMP, tissue inhibitor of metalloproteinase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; HPX domain, hemopexin-like domain; MTCBP-1, MT1-MMP cytoplasmic tail-binding protein-1; rMTCBP-1, recombinant MTCBP-1; ARD, aci-reductone dioxygenase; DSBH, double-stranded β helix; kb, kilobases; WT, wild type. is a member of the matrix metalloproteinase (MMP) family that collectively degrades most components of the extracellular matrix (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5113) Google Scholar). By anchoring to the plasma membrane through a transmembrane domain, MT1-MMP acts in the pericellular space on the cell surface. This property is particularly suitable for the degradation of extracellular matrix required for cellular functions such as migration, invasion, proliferation, and the regulation of cell morphology (2Seiki M. Koshikawa N. Yana I. Cancer Metastasis Rev. 2003; 22: 129-143Crossref PubMed Scopus (73) Google Scholar, 3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (191) Google Scholar). Because MT1-MMP is frequently expressed in malignant tumors, it is believed to play a major role in tumor invasion by degrading the extracellular matrix in the direction of cell migration and by processing of cell surface molecules (2Seiki M. Koshikawa N. Yana I. Cancer Metastasis Rev. 2003; 22: 129-143Crossref PubMed Scopus (73) Google Scholar, 3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (191) Google Scholar, 4Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2368) Google Scholar). As a member of the MMP family, MT1-MMP has a propeptide, a catalytic domain, hinge, and a hemopexin-like (HPX) domain starting from the N terminus, and this extracellular portion is linked to the membrane through the transmembrane domain, which follows a short cytoplasmic tail composed of 20 amino acids (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5113) Google Scholar, 2Seiki M. Koshikawa N. Yana I. Cancer Metastasis Rev. 2003; 22: 129-143Crossref PubMed Scopus (73) Google Scholar, 5Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3877) Google Scholar). As an invasion-promoting enzyme, the activity, localization, and turnover of MT1-MMP are tightly regulated during cell locomotion (3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (191) Google Scholar). For example, MT1-MMP localizes at the leading edge of migrating cells, and this localization is regulated through interaction between CD44 and the HPX domain (6Mori H. Tomari T. Koshikawa N. Kajita M. Itoh Y. Sato H. Tojo H. Yana I. Seiki M. EMBO J. 2002; 21: 3949-3959Crossref PubMed Scopus (279) Google Scholar). CD44 is a cell adhesion molecule that acts as a receptor for hyaluronan and mediates flexible adhesion to the provisional hyaluronan-rich matrix in the dynamic ruffling membrane area. Such adhesion is expected to be followed by a firmer adhesion through integrins to generate force for migration (3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (191) Google Scholar). At the migration edge MT1-MMP forms oligomers through the HPX domain with possible participation of the cytoplasmic tail, the hinge region, and extracellular matrix that binds to the molecule (7Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar, 8Lehti K. Lohi J. Juntunen M.M. Pei D. Keski-Oja J. J. Biol. Chem. 2002; 277: 8440-8448Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 10Tam E.M. Wu Y.I. Butler G.S. Stack M.S. Overall C.M. J. Biol. Chem. 2002; 277: 39005-39014Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Activation of proMMP-2 by MT1-MMP is expected to be carried out efficiently within the oligomeric complex at the leading edge (7Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar). MT1-MMP exposed on the cell surface is regulated negatively by TIMPs (5Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3877) Google Scholar), auto-degradation (11Maquoi E. Frankenne F. Baramova E. Munaut C. Sounni N.E. Remacle A. Noel A. Murphy G. Foidart J.M. J. Biol. Chem. 2000; 275: 11368-11378Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and internalization (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar, 13Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar). Internalization of MT1-MMP depends on the cytoplasmic tail, and an LLY motif in the region was found to act as a binding site for AP-2 complex that mediates incorporation of target proteins into clathrin-coated pits (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). Deletion of the cytoplasmic tail inhibits not only the internalization of MT1-MMP but also the invasion-promoting activity against the reconstituted basement membrane (Matrigel) mediated by MT1-MMP (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar, 14Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. J. Biol. Chem. 2000; 275: 15006-15013Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Thus, the proteolytic activity of MT1-MMP alone is not enough for the invasion-promoting activity, and it has to be regulated further by the function of the cytoplasmic tail. Although the cytoplasmic tail is short, containing 20 amino acids, it is reported to affect functions of MT1-MMP such as the formation of oligomers (8Lehti K. Lohi J. Juntunen M.M. Pei D. Keski-Oja J. J. Biol. Chem. 2002; 277: 8440-8448Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), localization of the enzyme to the proteolytically active protrusions (invadopodia) (15Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (363) Google Scholar), internalization (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar, 13Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar), and cell migration and invasion (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar, 14Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. J. Biol. Chem. 2000; 275: 15006-15013Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, it has not been elucidated how the cytoplasmic tail affects these functions except for the mechanism of internalization. To obtain clues about the mechanism by which the cytoplasmic tail regulates MT1-MMP, we attempted to isolate genes whose products interact with this portion using the yeast two-hybrid screening system. Using a cDNA library established from human fibroblast WI-38 cells we isolated a new gene whose product shows homology to members of the Cupin superfamily, a new family composed of proteins with diverse functions and which has a conserved three-dimensional structure (16Dunwell J.M. Culham A. Carter C.E. Sosa-Aguirre C.R. Good-enough P.W. Trends Biochem. Sci. 2001; 26: 740-746Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). We named this protein MT1-MMP cytoplasmic tail-binding protein-1 (MTCBP-1). MTCBP-1 is expressed as a 19-kDa protein and is co-localized with MT1-MMP at the adherent membrane edge. It was also found in the cytoplasm and nucleus. The formation of a complex between MTCBP-1 and MT1-MMP was confirmed by co-immunoprecipitation. Characteristics of MTCBP-1 and its possible role as an invasion suppressor are discussed. Yeast Two-hybrid Analysis—A LexA-based yeast two-hybrid screening was performed as described in the instructions for the MATCH-MAKER two-hybrid kit (Clontech) using the cytoplasmic tail sequence of MT1-MMP as bait and a galactose inducible prey-fusion library. A DNA fragment encoding the intracellular sequence of human MT1-MMP (Arg-563–Val-582) was amplified by PCR using primers (5′-ggaattcagacgccatgggacccccagg-3′ and 5′-gagctcgcctcagaccttgtccagcagggaac-3′) and ligated to the LexA-encoding sequence for expression as a fusion protein. The fragment was subcloned into the yeast expression vector pEG202. The basal transcription activation activity of the bait plasmid pLexA-MT1-MMP-tail was negligible. The MATCHMAKER cDNA library used for screening was generated from poly(A)+ RNA isolated from a human lung fibroblast cell line, WI-38 (Clontech). Potential interactors were screened by auxotrophic selection on plates supplemented with galactose or glucose but lacking histidine, leucine, tryptophan, and uracil (Gal/-HLTU or Glu/-HLTU) and for the ability to metabolize X-gal on Gal/X-gal/-HTU or Glu/X-gal/-HTU plates. Positive colonies that grew on Gal/-HLTU plates and appeared blue on Gal/X-gal/-HLTU plates were collected. Northern Blot Analysis—Northern blot analysis was performed using The Human Multiple Tissue Northern (MTNTM) blot membrane (Clontech). Fragments of the cDNAs for human MT1-MMP, MTCBP-1, and glyceraldehyde-3-phosphate dehydrogenase were labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Biosciences) and used as probes. Reverse Transcription (RT)-PCR—First-strand cDNA was synthesized from 3 μg of total RNA using 0.3 μg of each random primer (Invitrogen) and 200 units of Superscript II RNase H reverse transcriptase (RTase) (Invitrogen). After removal of the random primers, 1 μl of the RT product was used as a template for PCR (25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min). Primer sequences are indicated in the figure legends. Glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified similarly as an internal control. Cell Culture and Transfection—Human fetus lung normal diploid fibroblasts (WI-38 and TIG-20), human fibrosarcoma (HT1080), and green monkey kidney (COS-1) cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (Sigma). Human fibrosarcoma (SW684) cells were cultured at 37 °C in Leibovitz's L-15 medium (Sigma). All media were supplemented with 10% fetal bovine serum. Cells were seeded in 6-well plates at 1.0–1.5 × 105 cells/well, and transfection was carried out after 16 h using FuGENE 6™ (Roche Applied Science) according to the manufacturer's instructions. Immunoprecipitation—Transfected cells (1.5 × 105 cells) cultured in 6-well dishes were lysed in a lysis buffer (1% Brij-99, 50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1% deoxycholic acid, and 0.1% SDS) in the presence of a protease inhibitor mixture (Roche Applied Science). The cell lysate was clarified by centrifugation at 15,000 rpm for 15 min, and the supernatant was incubated with anti-FLAG M2 antibody-conjugated agarose beads (Sigma) for 2 h at 4 °C. After the beads were collected and washed three times with lysis buffer, precipitates were eluted using a FLAG peptide and then analyzed by Western blotting using the same antibody. Subcellular Fractionation—After washing, cells in a culture dish were collected with a cell scraper on ice and suspended in 1 ml of 10 mm Tris-buffer, pH 7.4, containing 250 mm sucrose and proteinase inhibitor mixture (Roche Applied Science). Then the cells were homogenized using a Dounce homogenizer (20 strokes) and centrifuged in a microtube at 1500 rpm for 10 min at 4 °C to remove the nucleus and undisrupted cells. The supernatant fraction was collected and centrifuged further at 65,000 rpm for 1 h at 4 °C using a Beckman rotor (NTV-90). Plasma membrane fraction was recovered in the pellet, and cytoplasmic proteins were in the supernatant. Western Blot Analysis—Human tissue samples (Protein Medley™) (Clontech) and cells in culture were lysed in a SDS sample buffer containing 2-mercaptoethanol. Proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). After the blocking of the membrane with 10% fat-free dry milk in Tris-buffered saline (20 mm Tris-HCl, pH 7.5, 150 mm NaCl), the membrane was probed with antibodies for detection. The membrane was further probed with alkaline phosphatase-conjugated anti-mouse IgG or anti-rabbit IgG (Sigma) to visualize the reacted antibody. Purification of Recombinant MTCBP-1 and Preparation of a Polyclonal Antibody—Recombinant human MTCBP-1 was expressed in the Escherichia coli strain BL21 (DE3) pLysS (Stratagene) by transfecting MTCBP-1/pRSET B vector plasmid (Invitrogen), with which expression of the gene can be induced by adding 0.4 mm isopropyl-1-thio-β-d-galactopyranoside to the culture medium. Cells were collected and sonicated in a TNC buffer (50 mm Tris-HCl, 150 mm NaCl, 10 mm CaCl2, and 0.02% NaN3) containing 2 mm phenylmethylsulfonyl fluoride. Supernatant was collected, and the His6-tagged protein was purified on a chelating Sepharose column. The bound protein was eluted with TNC buffer containing 500 mm imidazole and separated further with a gel filtration column of Sephacryl S-200 (Amersham Biosciences). To remove the His tag, EnterokinaseMax™ (Invitrogen) was used under the reaction conditions recommended by the manufacturer. To obtain polyclonal antibody for MTCBP-1 two female rabbits were immunized with the purified recombinant protein conjugate emulsified with an equal volume of Freund's complete adjuvant. A week after the last injection serum was obtained from the animals and subjected to a 40% saturated ammonium sulfate fractionation. Antibody reactive to MTCBP-1 was further purified using an affinity column of Sepharose 4B conjugated with recombinant MTCBP-1 (rMTCBP-1). Indirect Immunofluorescence Staining—Transfected HT-1080 cells were seeded on fibronectin/gelatin-coated coverslips. After 24 h the cells were washed with phosphate-buffered saline three times and fixed with 3% paraformaldehyde in phosphate-buffered saline. To detect the MTCBP-1 signal, cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. After blocking treatment with 5% goat serum and 3% bovine serum albumin in phosphate-buffered saline for 1 h at room temperature, cells were reacted with rabbit anti-MTCBP-1 antibody or mouse anti-FLAG M2 antibody at room temperature for 2 h. To visualize MTCBP-1 and FLAG-tagged MT1-MMP, cells were further incubated with Alexa™488-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., OR) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch laboratories, Inc.). Fluorescence signals were detected using a Bio-Rad MRC-1024 confocal laser microscope. Gelatin Zymography—Gelatin zymography was conducted with an SDS-polyacrylamide gel containing gelatin (0.8 mg/ml) as described previously (4Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2368) Google Scholar). The samples were mixed with SDS/PAGE loading buffer without a reducing agent and subjected to electrophoretic analysis at room temperature. Enzyme activity was visualized as negative staining with Coomassie Brilliant Blue R-250. Phagokinetic Track Motility Assay—The phagokinetic track motility assay was performed as described previously (12Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). Colloidal gold-coated coverslips were placed in a 12-well plate, and transfected cells were seeded at 3 × 103 cells/well. After 12 h of incubation at 37 °C, the phagokinetic tracks were visualized under bright-field illumination using a CoolSNAP-fx monochrome CCD camera (Roper Scientific). The track area was measured using NIH Image software Version 1.62. Matrigel Invasion Assay—The Matrigel invasion assay was performed as described previously (7Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar) according to the manufacturer's instructions (BD Biosciences). MTCBP-1 plasmid was introduced into HT-1080 cells with a plasmid for the expression of green fluorescent protein. The transfected cells were suspended in Dulbecco's modified Eagle's medium without serum and seeded onto a Matrigel-coated filter (8-μm pore) in the Transwell chamber. Fetal bovine serum (10%) was added to the medium in the lower chambers and incubated for 12 h at 37 °C. Non-invading cells remaining on the upper surface of the filter were removed and fixed with 3% paraformaldehyde. The green fluorescent protein-positive cells on the lower surface of the filter were enumerated under microscope at a magnification of ×400. Each assay was performed in triplicate, and five microscopic fields from each of the three filters were counted. Screening of Human Genes Whose Products Bind MT1-MMP at the Cytoplasmic Tail—To isolate candidate genes whose products bind the cytoplasmic tail of MT1-MMP, the yeast two-hybrid system was used to screen a human cDNA library established from lung fibroblast cells (WI-38). The cytoplasmic tail peptide (RRHGTPRRLLYCQRSLLDKV) was expressed as bait fused to the DNA binding domain of LexA protein. From 8 × 107 transformants, 23 clones were obtained and confirmed to be true positives. These clones can be divided into two groups by their size (1062 and 1617 bp) with one exception. DNA sequence analysis revealed that the 1062-bp fragment matched exactly the 5′ portion of the 1617-bp fragment, sharing the same 5′ end, and that both had a poly(A) tail at their 3′ end (Fig. 1). Thus, these two cDNAs are presumably derived from transcripts of a single gene that has multiple transcriptional termination sites. Both transcripts have one open reading frame that potentially encodes a 179-amino acid polypeptide. Because the product of the gene is expected to bind to the cytoplasmic tail of MT1-MMP, it was named MT1-MMP cytoplasmic-binding protein-1 (MTCBP-1). One cDNA that did not show any homology to the MTCBP-1 gene was not analyzed further in this study. Structure of MTCBP-1 and Its Homologues—A BLAST search with MTCBP-1 of the human DNA databases yielded a gene called Sip-L (Fig. 2), the expression of which is reported to render cells susceptible to infection by human hepatitis C virus by allowing its replication (17Yeh C.T. Lai H.Y. Chen T.C. Chu C.M. Liaw Y.F. J. Virol. 2001; 75: 11017-11024Crossref PubMed Scopus (29) Google Scholar). Although the two genes matched completely, the reported Sip-L cDNA lacks the 5′ sequence of MTCBP-1 that includes the non-coding and part of the 5′-coding sequence. As a result, Sip-L lacks the N-terminal 63 amino acids of MTCBP-1. However, the transcripts and protein product of Sip-L have not been characterized well in the previous study (17Yeh C.T. Lai H.Y. Chen T.C. Chu C.M. Liaw Y.F. J. Virol. 2001; 75: 11017-11024Crossref PubMed Scopus (29) Google Scholar) as discussed later. MTCBP-1 also showed significant homology to the bacterial genes that encode aci-reductone dioxygenase (ARD) used in the salvage pathway of methionine metabolism. The homology of MTCBP-1 to the ARD of Klebsiella oxytoca (28.2%) and other homologues in different species is summarized in Fig. 2B. MTCBP-1 homologues are composed of 178–188 amino acids. Among them, ARDs of Klebsiella pneumoniae and oxytoca, including the three-dimensional structure, are the best characterized (18Dai Y. Wensink P.C. Abeles R.H. J. Biol. Chem. 1999; 274: 1193-1195Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 19Dai Y. Pochapsky T.C. Abeles R.H. Biochemistry. 2001; 40: 6379-6387Crossref PubMed Scopus (143) Google Scholar, 20Pochapsky T.C. Pochapsky S.S. Ju T. Mo H. Al-Mjeni F. Maroney M.J. Nat. Struct. Biol. 2002; 9: 966-972Crossref PubMed Scopus (87) Google Scholar), although the functions of the eukaryotic homologues have yet to be studied. ARD has a β-barrel hold that is also known as a double stranded β helix (DSBH) domain (20Pochapsky T.C. Pochapsky S.S. Ju T. Mo H. Al-Mjeni F. Maroney M.J. Nat. Struct. Biol. 2002; 9: 966-972Crossref PubMed Scopus (87) Google Scholar) and is characteristically conserved in members of the Cupin superfamily that was proposed recently (16Dunwell J.M. Culham A. Carter C.E. Sosa-Aguirre C.R. Good-enough P.W. Trends Biochem. Sci. 2001; 26: 740-746Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Despite this structural conservation, the members of this family vary widely in function and include metabolic enzymes, transcription factors, scaffold proteins etc. Interaction between MTCBP-1 and MT1-MMP in Cells— rMTCBP-1 was expressed in E. coli and purified as described under “Materials and Methods.” Rabbit polyclonal antibody was also prepared using rMTCBP-1 as an immunogen. To examine whether MTCBP-1 binds to the cytoplasmic tail of MT1-MMP directly, a cytoplasmic tail peptide with His6 tag (CP-WT) and a control peptide with a randomized sequence (CP-RD) were prepared. The peptides were immobilized on a nitrocellulose membrane filter and then blotted with rMTCBP-1. The protein bound to the filter was visualized using anti-MTCBP-1 antibody conjugated with alkaline phosphatase (Fig. 3A). MTCBP-1 was bound to CP-WT but not CP-RD. The binding to CP-WT was specific because it was blocked almost completely with an excess amount of the CP-WT peptide but not with CP-RD. To confirm the binding within cells either a full-length MT1-MMP having a FLAG tag immediately downstream of the furin site (MT1F) or its mutant lacking the cytoplasmic tail (dCPF) was expressed together with MTCBP-1 in human fibrosarcoma HT1080 cells. Transfected cells were lysed, and MT1F was immunoprecipitated using anti-FLAG antibody. The precipitates were eluted from the antibody using FLAG peptide, and the eluate was analyzed further by Western blotting using anti-MTCBP-1 antibody (Fig. 3B). Expression of each protein in the cells was confirmed by Western blotting (Fig. 3B, total lysate). Although MTCBP-1 itself was not precipitated by the anti-FLAG antibody, it was precipitated with MT1F when both were co-expressed. The mutant MT1F lacking the cytoplasmic tail (dCPF) failed to precipitate MTCBP-1. Thus, MTCBP-1 binds to the cytoplasmic tail of MT1-MMP in the cells. MT1-MMP Recruits MTCBP-1 to the Plasma Membrane Fraction—If MTCBP-1 bound MT1-MMP, the proteins would co-localize in the cells. MT1-F and MTCBP-1 were co-expressed in HT1080 cells, and localization of the products was examined by immunostaining (Fig. 3C). MT1F was detected on the cell surface especially at the ruffling edge when the cells were examined without permeabilization (no treatment with Triton X-100). Under the same conditions MTCBP-1 was not detected as it is a cytoplasmic protein. After the cells were treated with Triton X-100, co-localization of MTCBP-1 and MT1F was detected at the periphery of the cells (Fig. 3C). Some MT1F signals within the cells presumably represent the translation products in vesicles and the Golgi apparatus. Although MTCBP-1 was also detected in the cytoplasm, its localization did not coincide with that of MT1F. Some MTCBP-1 was detected in the nucleus as well, indicating the possibility that MTCBP-1 shuttles between the three compartments, membrane, cytoplasm, and nucleus. To confirm the interaction of both proteins further, plasma membrane-enriched fraction was prepared and examined by Western blotting (Fig. 3D). Accumulation of MTCBP-1 in the membrane fraction was not much when it is expressed without MT1F. However, co-expression increased the amount of MTCBP-1 in the membrane fraction significantly. This accumulation was dependent on the cytoplasmic tail of MT1F because co-expression of dCPF failed to increase this amount. Appropriate fractionation was monitored using marker proteins such as transferring receptor (TfnR) for membrane proteins and actin for cytoplasmic proteins. Expression of MT1F and dCPF did not affect the expression levels of MTCBP-1 and the fractionation (Fig. 3D). Thus, the results strongly suggest that MT1-MMP forms a complex with MTCBP-1 within the cells. Expression and Tissue Distribution of MTCBP-1—The expression of MTCBP-1 mRNA in different human tissues was examined by Northern blotting and compared with that of MT1-MMP (Fig. 4A). Three MTCBP-1 transcripts (1.2, 1.5, and 2.0 kb) were detected in tissue samples. The three presumably represent transcripts terminating at different sites. Corresponding to the three transcripts, three potential poly(A) signals can be seen in the 3′-non-coding region of the 1.6-kb cDNA sequence as indicated in Fig. 1. The expression levels were higher in heart and lower in brain and placenta (Fig. 4A). On the other hand, MT1-MMP mRNA was predominantly expressed in lung and placenta, where MTCBP-1 expression is low. Thus, MT1-MMP may not require MTCBP-1 to function, at least in these tissues. The antibody against MTCBP-1 detected a 19-kDa protein in the COS-1 cells transfected with the expression plasmid for MTCBP-1 but not in the mock-transfected cells (Fig. 4B). A band similar in size was also detected in the tissue extract from human lung and liver. Inverse Correlation of MTCBP-1 and MT1-MMP Expression in the Transformed and Non-transformed Human Cell Lines— MT1-MMP is frequently expressed in human tumors and tumor-derived cell lines. Thus, expression of MTCBP-1 and MT1-MMP was examined further using cell lines to confirm the relationship between the two (Fig. 5, A and B). As tumor cell lines, four gastric carcinoma (MKN-7, MKN-28, NUGU-3, and NUGC-4) and two fibrosarcoma (HT1080 and SW684) cell lines were analyzed by RT-PCR using specific primers (Fig. 5A). Non-transformed fibroblasts WI-38 and TIG-20 were also examined. MT1-MMP mRNA was detected in the tumor cell lines, reported at higher levels than in the non-transformed cells (Fig. 5B). In contrast, althoug