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Title: $Methionine Aminopeptidase 2 Is a New Target for the Metastasis-associated Protein, S100A4
Abstract: S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis. S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis. methionine aminopeptidase 2 α-minimal essential medium fetal bovine serum basic fibroblast growth factor hemagglutinin glutathione S-transferase Dulbecco's phosphate-buffered saline protection of eIF2α phosphorylation tetramethylrhodamine isothiocyanate Calcium-binding proteins transduce intracellular calcium signals to many biological processes including protein phosphorylation, enzyme activities, cell proliferation and differentiation, dynamics of cytoskeletal organization and Ca2+ homeostasis, and regulate the cytosolic Ca2+ level (1Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2285) Google Scholar). S100 proteins are structurally related to the well known EF-hand calcium-binding proteins, such as calmodulin and troponin C, and comprise 19 members that are differentially expressed in a variety of cell types (2Donato R. Biochim. Biophys. Acta. 1999; 1450: 191-231Crossref PubMed Scopus (602) Google Scholar, 3Donato R. Int. J. Biochem. 2001; 33: 638-668Google Scholar, 4Schaefer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Crossref PubMed Scopus (1087) Google Scholar). S100A4, one member of the S100 protein family, is a small acidic protein consisting of 101 amino acid residues (2Donato R. Biochim. Biophys. Acta. 1999; 1450: 191-231Crossref PubMed Scopus (602) Google Scholar, 3Donato R. Int. J. Biochem. 2001; 33: 638-668Google Scholar, 4Schaefer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Crossref PubMed Scopus (1087) Google Scholar). Expression of theS100A4 gene (also known as pEL98/mts1/p9Ka/18A2/calvasculin/CAPL) has been implicated in cell cycle progression, cell differentiation, and neoplastic progression (2Donato R. Biochim. Biophys. Acta. 1999; 1450: 191-231Crossref PubMed Scopus (602) Google Scholar, 3Donato R. Int. J. Biochem. 2001; 33: 638-668Google Scholar, 4Schaefer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Crossref PubMed Scopus (1087) Google Scholar). In particular, the observations that S100A4 is involved in tumor cell invasion and metastasis have attracted attention. It has been shown that S100A4 is highly expressed in different rodent invasive and metastatic tumor cells (5Ebralidze A. Tulchinsky E. Grigorian M. Afanasyeva A. Senin V. Revazova E. Lukanidin E.M. Genes Dev. 1989; 3: 1086-1093Crossref PubMed Scopus (320) Google Scholar, 6Takenaga K. Nakamura Y. Endo H. Sakiyama S. Jpn. J. Cancer Res. 1994; 85: 831-839Crossref PubMed Scopus (88) Google Scholar) and that manipulation of the expression of S100A4 in tumor cells results in the alteration of their invasive and metastatic potential (7Davies B.R. Davies M.P.A. Gibbs F.E.M. Barraclough R. Rudland P.S. Oncogene. 1993; 8: 999-1008PubMed Google Scholar, 8Parker C. Whittaker P.A. Usmani B.A. Lakshimi M.S. Sherbet G.V. DNA Cell Biol. 1994; 13: 1021-1028Crossref PubMed Scopus (53) Google Scholar, 9Grigorian M.S. Tulchinsky E.M. Zain S. Ebralidze A.K. Kramerov D.A. Kriajevska M.V. Georgiev G.P. Lukanidin E.M. Gene (Amst.). 1993; 135: 229-238Crossref PubMed Scopus (100) Google Scholar, 10Takenaga K. Nakamura Y. Sakiyama S. Oncogene. 1997; 14: 331-337Crossref PubMed Scopus (142) Google Scholar). In addition, we and others have recently demonstrated that the elevated levels of S100A4 are associated with the more malignant phenotype and prognosis in human breast and colorectal adenocarcinoma specimens (11Takenaga K. Nakanishi H. Wada K. Suzuki M. Matsuzaki O. Matsuura A. Endo H. Clin. Cancer Res. 1997; 3: 2309-2316PubMed Google Scholar,12Rudland P.S. Platt-Higgins A. Renshaw C. West C.R. Winstanley J.H. Robertson L. Barraclough R. Cancer Res. 2000; 60: 1595-1603PubMed Google Scholar). Although the precise mechanisms by which S100A4 affects the invasive and metastatic phenotypes of tumor cells remain to be solved, the expression level of S100A4 has been shown to correlate positively with the expression of matrix metalloproteinases (13Bjornland K. Winberg J.O. Odegaard O.T. Hovig E. Loennechen T. Aasen A.O. Fodstad O. Maelandsmo G.M. Cancer Res. 1999; 59: 4702-4708PubMed Google Scholar) and cell motility (6Takenaga K. Nakamura Y. Endo H. Sakiyama S. Jpn. J. Cancer Res. 1994; 85: 831-839Crossref PubMed Scopus (88) Google Scholar, 14Ford H.L. Salim M.M. Chakravarty R. Aluiddin V. Zain S.B. Oncogene. 1995; 11: 2067-2075PubMed Google Scholar) and negatively with the expression of E-cadherin (15Yonemura Y. Endou Y. Kimura K. Fushida S. Bandou E. Taniguchi K. Kinoshita K. Ninomiya I. Sugiyama K. Heizmann C.W. Schaefer B.W. Sasaki T. Clin. Cancer Res. 2000; 6: 4234-4242PubMed Google Scholar, 16Keirsebilck A. Bonne S. Bruyneel E. Vermassen P. Lukanidin E. Mareel M. van Roy F. Cancer Res. 1998; 58: 4587-4591PubMed Google Scholar). S100A4 is also expressed in a variety of normal cells including lymphocytes and vascular endothelial cells (17Takenaga K. Nakamura Y. Sakiyama S. Cell Struct. Funct. 1994; 19: 133-141Crossref PubMed Scopus (55) Google Scholar, 18Gibbs F.E.M. Barraclough R. Platt-Higgins A. Rudland P.S. Wilkinson M.C. Parry E.W. J. Histochem. Cytochem. 1995; 43: 169-180Crossref PubMed Scopus (59) Google Scholar). A promising approach to gain insight into the functions of this protein might be studies on its target proteins. Indeed, nonmuscle tropomyosin (19Takenaga K. Nakamura Y. Sakiyama S. Hasegawa Y. Sato K. Endo H. J. Cell Biol. 1994; 124: 757-768Crossref PubMed Scopus (157) Google Scholar), nonmuscle myosin heavy chain (20Kriajevska M.V. Cardenas M.N. Grigorian M.S. Ambartsumian N.S. Georgiev G.P. Lukanidin E.M. J. Biol. Chem. 1994; 269: 19679-19682Abstract Full Text PDF PubMed Google Scholar, 21Ford H.L. Zain S.B. Oncogene. 1995; 10: 1597-1605PubMed Google Scholar), and F-actin (22Watanabe Y. Usada N. Minami H. Morita T. Tugane S. Ishikawa R. Kohama K. Tomida Y. Hidaka H. FEBS Lett. 1993; 324: 51-55Crossref PubMed Scopus (82) Google Scholar) have been reported as the targets, suggesting a possible participation of S100A4 in the regulation of cytoskeletal dynamics and cell motility (6Takenaga K. Nakamura Y. Endo H. Sakiyama S. Jpn. J. Cancer Res. 1994; 85: 831-839Crossref PubMed Scopus (88) Google Scholar, 14Ford H.L. Salim M.M. Chakravarty R. Aluiddin V. Zain S.B. Oncogene. 1995; 11: 2067-2075PubMed Google Scholar). More recently, two other proteins, S100A1 (23Wang G. Rudland P.S. White M.R. Barraclough R. J. Biol. Chem. 2000; 275: 11141-11146Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and tumor suppressor p53 protein (24Grigorian M. Andresen S. Tulchinsky E. Kriajevska M. Carlberg C. Kruse C. Cohn M. Ambartsumian N. Christensen A. Selivanova G. Lukanidin E. J. Biol. Chem. 2001; 276: 22699-22708Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), have been demonstrated to interact physically and functionally with S100A4. Methionine aminopeptidase 2 (MetAP2)1 is one of two enzymes that catalyze the co-translational removal of the initiator methionine residue from nascent peptides in eukaryotes (25Bradshaw R.A. Brickey W.W. Walker K.W. Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). In addition to its methionine aminopeptidase activity, MetAP2 is shown to regulate protein synthesis by protecting the α subunit of eukaryotic initiation factor-2 (eIF2) from phosphorylation (POEP activity) (26Datta B. Ray M.K. Chakrabarti D. Wylie D.E. Gupta N.K. J. Biol. Chem. 1989; 264: 20620-20624Abstract Full Text PDF PubMed Google Scholar,27Ray M.K. Datta B. Chakraborty A. Chattopadyay A. Meza-Keuten S. Gupta N.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 539-543Crossref PubMed Scopus (88) Google Scholar). Recently, MetAP2 has been identified as a physiologically relevant target for potent angiogenesis inhibitors, fumagillin and ovalicin (28Sin N. Meng L. Wang M.Q. Wen J.J. Bornmann W.G. Crews C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6099-6103Crossref PubMed Scopus (602) Google Scholar,29Griffith E.C., Su, Z. Turk B.E. Chen C. Chang Y-H., Wu, Z. Biemann K. Liu J.O. Chem. Biol. 1997; 4: 461-471Abstract Full Text PDF PubMed Scopus (407) Google Scholar). A synthetic analog of fumagillin, TNP-470, selectively and covalently binds to MetAP2 and blocks its aminopeptidase activity, but not its POEP activity, at the comparable concentrations required for inhibition of endothelial cell growth (30Turk B.E. Griffith E.C. Wolf S. Biemann K. Chang Y-H. Liu J.O. Chem. Biol. 1999; 6: 823-833Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Interestingly, the growth of endothelial cells is much more sensitive to this compound than that of tumor cells (31Kusaka M. Sudo K. Matsutani E. Kozai Y. Marui S. Fujita T. Ingber D. Folkman J. Br. J. Cancer. 1994; 69: 212-216Crossref PubMed Scopus (233) Google Scholar, 32Yanase T. Tamura M. Fujita K. Kodama S. Tanaka K. Cancer Res. 1993; 53: 2566-2570PubMed Google Scholar). MetAP2 is thus thought to concern endothelial cell proliferation through N-terminal methionine processing of as yet unknown proteins essential for endothelial cell growth (30Turk B.E. Griffith E.C. Wolf S. Biemann K. Chang Y-H. Liu J.O. Chem. Biol. 1999; 6: 823-833Abstract Full Text PDF PubMed Scopus (100) Google Scholar). In an effort to identify additional target proteins for S100A4, we employed the yeast two-hybrid system and screened a cDNA library with S100A4 cDNA as bait. Here, we report that MetAP2 is a new target for S100A4. Mouse endothelial cell line MSS31 (a gift of Dr. N. Yanai, Tohoku University) (33Yanai N. Satoh T. Obinata M. Cell Struct. Funct. 1991; 16: 87-93Crossref PubMed Scopus (68) Google Scholar) was routinely cultured in α-MEM supplemented with 10% FBS. When they were treated with bFGF, DNA synthesis, cell migration, and tube formation on type I collagen gels were induced as described previously (data not shown) (34Tanaka K. Abe M. Sato Y. Jpn. J. Cancer Res. 1999; 90: 647-654Crossref PubMed Scopus (121) Google Scholar). Human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. For screening a mouse T cell lymphoma cDNA library constructed with pACT, a yeast vector carrying the GAL4-activation domain and Leu selectable marker (CLONTECH), a bait plasmid was constructed by subcloning the mouse S100A4 coding region excised from the pEL98 plasmid (35Goto K. Endo H. Fujiyoshi T. J. Biochem. 1988; 103: 48-53Crossref PubMed Scopus (76) Google Scholar) into pBTM116, a yeast vector containing the LexA domain and a Trp selectable marker (36Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (306) Google Scholar). The screening of the library was performed essentially according to the lithium yeast transformation protocol (37Vojtek A.B. Hollenberg S.M. Methods Enzymol. 1995; 255: 331-342Crossref PubMed Scopus (244) Google Scholar). Briefly, L40 yeast strain cells (Invitrogen) were transformed first with the bait plasmid and then selected on tryptophan-free plates to produce a stable L40-LexA-S100A4 strain. A culture of the strain was then transformed with the pACT/cDNA library and selected on leucine-, tryptophan-, and histidine-free plates and media. The transformants thus appeared were applied to a β-galactosidase colony-lift filter assay and the colonies that gave a blue color within 16 h were isolated. Both the bait and target plasmids were isolated from each individual clone. The recovered target plasmids were co-transformed into L40 cells along with the original bait plasmid LexA-S100A4, and the colony-lift assays were carried out to reconfirm the interaction in the yeast cells. A full-lengthMetAP2 cDNA was isolated by screening a mouse lung carcinoma cDNA library with the 5′-half of the MetAP2 coding sequence identified in the present study as a probe (GenBankTM accession number AF434712). Expression vectors of various truncated forms of MetAP2 were constructed using pcDNA3/HA3 vector (a gift of Dr. I Nishimoto, Keio University) and PCR-amplified cDNA fragments. The nucleotide sequences of the fragments were those corresponding to amino acids Met1-Ala229 (MetAP2-(1–229)), Met1-Pro109 (MetAP2-(1–109)), Lys110-Ala229 (MetAP2-(110–229)), Lys110-Phe169 (MetAP2-(110–169)), and Arg170-Ala229 (MetAP2-(170–229)). The following 5′ and 3′ primers carrying EcoRI andXhoI recognition sites at the respective 5′ end were used: MetAP2-(1–229)/5′-primer, 5′-AATGAATTCATGGCGGGCGTGGAGCAGGCA-3′; MetAP2-(1–229)/3′-primer, 5′-AATCTCGAGTTAAGCACGGTTGTTGAGAGAACCC-3′; MetAP2-(1–109)/5′-primer, 5′-AATGAATTCATGGCGGGCGTGGAGCAGGCA-3′; MetAP2-(1–109)/3′-primer, 5′-ATCTCGAGTTAGGCGGCTTCCTCGGCCGTGC-3′; MetAP2-(110–229)/5′-primer, 5′-ATGAATTCCCAAAAGTTCAAACAGACCCTCCC-3′; MetAP2-(110–229)/3′-primer, 5′-AATCTCGAGTTAAGCACGGTTGTTGAGAGAACACCC-3′; MetAP2-(110–169)/5′-primer, 5′-ATGAATCCCAAAAGTTCCAGACCCTCCC-3′; MetAP2-(110–169)/3′-primer, 5′-TATCTCGAGTTAGAAGTCGTTCCAGATCTC-3′; MetAP2-(170–229)/5′-primer, 5′-ATAGAATTCCGAGAAGCTGCGGTTGTTGAGGCACAT-3′; and MetAP2-(170–229)/3′-primer, 5′-AATCTCGAGTTAAGCACGGTTGTTGAGAGAACACCC-3′. The resulting PCR products were digested with EcoRI and XhoI, gel-purified, and then cloned into the EcoRI/XhoI site of a pcDNA3/HA3 vector. The entire coding sequence of theMetAP2 cDNA was also inserted in-frame in the pcDNA3/HA3 vector. The coding sequence of pEL98 (mouse S100A4) was amplified by PCR using the 5′ primer having a XhoI recognition site at the 5′ end, 5′-AGACTCGAGTCAACGGTTACCATGGCAAC-3′ and the 3′ primer having the NotI recognition sequence at the 5′ end, 5′-ATTGCGGCCGCGAGGAGTCTTCACTTCTTCC-3′. The resulting PCR fragments were digested with XhoI and NotI, gel-purified, and then cloned into NotI/XhoI cut pME18S/FLAG vector (a gift of Dr. K. Maruyama, Tokyo Medical and Dental University). The pEL98 cDNA clone and preparation of the pGEX/pEL98 plasmid expressing the GST/S100A4 fusion protein has been described previously (17Takenaga K. Nakamura Y. Sakiyama S. Cell Struct. Funct. 1994; 19: 133-141Crossref PubMed Scopus (55) Google Scholar, 35Goto K. Endo H. Fujiyoshi T. J. Biochem. 1988; 103: 48-53Crossref PubMed Scopus (76) Google Scholar). The coding sequences of human S100A1, S100A4, S100A6, and calmodulin were amplified by reverse transcription-PCR using total RNA prepared from human brain (CLONTECH) for S100A1, and from HeLa cells for S100A4, S100A6, and calmodulin. The respective forward and reverse primer pairs harboring EcoRI and XhoI recognition or EcoRV and XhoI recognition sites at the 5′ end were: 5′-GAATTCATGGGCTCTGAGCTGGAG-3′ and 5′-CTCGAGTCAACTGTTCTCCCAGAA-3′ for S100A1; 5′-GAATTCATGGCGTGCCCTCTGGAG-3′ and 5′-CTCGAGTCATTTCTTCCTGGGCTG-3′ for S100A4; 5′-GAATCCATGGCATGCCCCCTGGAT-3′ and 5′-CTCGAGTCAGCCCTTGAGGGCTTC-3′ for S100A6; and 5′-GCGGATATCAAATGGCTGATCAGCTGACCGAA-3′ and 5′-GGCTCGAGTCATTTTGCAGTCATCATCTGTACG-3′ for calmodulin. For preparation of the plasmid expressing GST fused to human MetAP2-(224–478), first strand cDNA was synthesized from total RNA extracted from peripheral blood mononuclear cells by the use of the T-primed first strand cDNA synthesis kit (Ready to Go, AmershamBiosciences). The nucleotide sequence corresponding to amino acid residues 224–478 of MetAP2 was PCR-amplified from the first strand cDNA using the following primer pairs: 5′-GCCGAATTCTCTCTCAATAATTGTGCTGCCC-3′ and 5′-GAGCTCGAGTTAATAGTCATCTCCTCTGCTGC-3′. The resulting PCR fragments were digested with EcoRI and XhoI, except for calmodulin, which was treated with EcoRV andXhoI, gel-purified, and then cloned intoEcoRI/XhoI or EcoRV/XhoI cut pGEX/5X-1 vectors. Competent Escherichia coli JM109 cells were transformed with each of the pGEX recombinant plasmids. Expression of the GST fusion proteins was induced by 100 μm isopropyl-1-thio-β-d-galactoside for 3 h. The bacteria were pelleted by centrifugation, washed once with cold Dulbecco's phosphate-buffered saline (DPBS), and then resuspended in 25 mm Hepes (pH 7.6), 300 mmKCl, 1 mm EGTA, 12.5 mm MgCl2, 0.5% Triton X-100, 10% glycerol, 1 mm dithiothreitol, and appropriate amounts of protease inhibitor mixture (Sigma). After adding 500 μg/ml lysozyme, bacteria were incubated on ice for 20 min, sonicated, and then centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was removed and loaded onto a column of glutathione-Sepharose 4B (Amersham Biosciences). GST fusion proteins were eluted by 10 mm glutathione in 50 mmTris-HCl (pH 9.5), dialyzed against 1 mm CaCl2, 50 mm Tris-HCl (pH 7.5), and 150 mm NaCl. The GST/MetAP2-(224–478) fusion protein was separated on SDS-PAGE, eluted into 0.1% SDS, 25 mm Tris, 192 mmglycine, and applied to gel filtration using Sephadex G-50 medium (Amersham Biosciences) equilibrated with 0.1% SDS, 1% methanol. After lyophilization of the fraction, the purified fusion protein was dissolved in distilled water, dialyzed against 0.1% SDS, and used as an immunogen to generate anti-MetAP2 antibody. Anti-human MetAP2 antiserum was obtained after immunizing a rabbit with the fusion protein. The immunoglobulins in the immune serum were partially purified with ammonium sulfate precipitation (50%), dissolved in DPBS, extensively dialyzed against DPBS, and then passed through a GST-Sepharose column three times to absorb anti-GST antibody. The anti-MetAP2 antibody was affinity purified using the GST/MetAP2-(224–478) fusion protein immobilized onto the nitrocellulose membrane (38Cox S.V. Schenk E.A. Olmsted J.B. Cell. 1983; 35: 331-339Abstract Full Text PDF PubMed Scopus (80) Google Scholar). In vitro transcription and translation of genes subcloned into pcDNA3/HA3 was carried out with the TnT quick coupled transcription/translation system (Promega) using [35S]methionine as a tracer amino acid. The35S-labeled translation products were incubated with 10 μg of GST, GST/S100A4, or GST/calmodulin fusion proteins in buffer containing 0.5% (v/v) Nonidet P-40, 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride and either 1 mm CaCl2 or 1 mm EDTA at 4 °C for 60 min and then followed by the addition of glutathione-Sepharose 4B beads. After incubation for a further 60 min at 4 °C, the beads were washed five times with the above buffer and boiled for 3 min in SDS gel sample buffer. The released materials were subjected to SDS-PAGE. The gels were fixed and treated with ENLIGHTNING (PerkinElmer Life Sciences) before they were dried and exposed to Fuji RX medical x-ray film with an intensifying screen at −80 °C. Protein concentration was determined by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223660) Google Scholar) using bovine serum albumin as a standard. 293 cells were transfected with 5 μg of pcDNA3/HA3-J6-2 or pME18S/FLAG-S100A4 either alone or both using a Lipofectin (Invitrogen) method. After a 60-h incubation, cells were washed with DPBS, lysed with 0.5% Nonidet P-40, 50 mm Tris-HCl (pH 7.4), 150 mmNaCl, 1 mm phenylmethylsulfonyl fluoride, and 1 mm CaCl2 and centrifuged at 10,000 ×g. The resulting supernatants were collected and used for subsequent immunoprecipitation analyses. For in vivocross-linking experiments, cells were incubated with 1 mmdithiobis(succinimidyl propionate) (Sigma) in DPBS for 20 min at room temperature before cell lysis. After preclearing with protein A-Sepharose, cell lysates were incubated with a rabbit anti-S100A4 antibody (17Takenaga K. Nakamura Y. Sakiyama S. Cell Struct. Funct. 1994; 19: 133-141Crossref PubMed Scopus (55) Google Scholar) for 1 h at 4 °C and then with Protein A-Sepharose 4B beads for 1 h at 4 °C. The immunoprecipitates were resolved by SDS-PAGE using a 12.5% separating gel and blotted onto nitrocellulose filter. Immunoblot analysis was performed using a rat monoclonal anti-HA antibody (clone 3F-10, Roche Molecular Biochemicals), a mouse monoclonal anti-FLAG antibody (clone M2, Sigma), the corresponding horseradish peroxidase-labeled secondary antibodies, and an ECL Western blotting detection kit (Amersham Biosciences). Recombinant His-tagged human MetAP2 was expressed in Sf9 cells by using baculovirus (a gift of Dr. J. O. Liu, Massachusetts Institute of Technology). The cells were harvested 36 h after baculovirus infection and lysed in 5 packed cell volumes of cold buffer B (10 mm Hepes, pH 8.0, 100 mm KCl, 1.5 mm MgCl2, 10% glycerol) containing 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 μg/ml pepstatin. The cell lysate was incubated on ice for 10 min and centrifuged at 10,000 × g for 10 min. The supernatant was diluted in buffer B and incubated for 1 h at 4 °C with pre-equilibrated Talon resin (CLONTECH). After extensive washing with buffer B, the resin was slurried into a Bio-Rad Econo column. The recombinant MetAP2 thus made was eluted with buffer B containing 50 mmimidazole. The fractions containing the highest amounts of MetAP2 were pooled and dialyzed against buffer B. The purity of recombinant MetAP2 was confirmed by SDS-PAGE and immunoblotting. Total RNA was extracted from MSS31 cells and the mouse heart using the RNeasy kit (Qiagen). Twenty μg of total RNA were electrophoresed on a 1% agarose gel containing formaldehyde and transferred to nylon filters. Blots were hybridized with 32P-labeled mouseS100A1, S100A4, S100A6, orMetAP2 cDNA that was prepared by the random primer method. Filters were finally washed at 50 °C in 30 mmNaCl, 3 mm sodium citrate, and 0.1% SDS. MSS31 cells were serum starved in α-MEM containing 0.1% FBS for 26 h and treated with 20 ng/ml bFGF for the following 16 h. Untreated and bFGF-treated cells were washed and lysed in 150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 1% Triton X-100, 1 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride. Cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C and the supernatant was used for subsequent analyses. SDS-PAGE was carried out using 12.5% acrylamide gels. Immunoblot analysis was performed using anti-S100A4 antibody, anti-MetAP2 antibody, anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (CHEMICON International, Inc.), and the corresponding secondary antibodies as described above. MSS31 cells maintained on coverslips in α-MEM containing 0.1% FBS for 26 h were treated with 20 ng/ml bFGF for 16 h. The cells were washed three times with DPBS and fixed for 30 min with 4% formaldehyde and 5% sucrose in DPBS. After permeabilizing with 0.5% Triton X-100 in DPBS for 4 min, the cells were treated with 3% bovine serum albumin in DPBS containing 0.1% glycine for 1 h to block nonspecific binding sites. After washing with DPBS, the cells were incubated for 1 h with a mixture of affinity purified rabbit anti-MetAP2 antibodies and rat anti-S100A4 monoclonal antibody (clone 4A2) (17Takenaga K. Nakamura Y. Sakiyama S. Cell Struct. Funct. 1994; 19: 133-141Crossref PubMed Scopus (55) Google Scholar), rinsed, and simultaneously stained with a mixture of TRITC/goat anti-rabbit IgG and fluorescein isothiocyanate/goat anti-rat IgG. After rinsing with DPBS, the coverslips were mounted in 50% glycerol in DPBS containing 1 mg/mlp-phenylenediamine to inhibit photobleaching. The cells were observed under a confocal laser scanning microscope (Fluoview, Olympus, Japan). To explore a new type of target proteins capable of interacting with S100A4, we adopted the yeast two-hybrid system and screened a mouse T cell lymphoma cDNA library with mouseS100A4 cDNA as the bait. After β-galactosidase colony lift filter assays, positive colonies were picked up and target plasmids were recovered from each of them. The purified plasmid DNAs were again co-transformed individually into L40 yeast cells along with the S100A4 bait plasmid. After repeating the procedure twice, we finally obtained 27 positive transformants in the colony lift filter assays. Plasmid DNA in each transformant was isolated and the cDNA inserts were subjected to nucleotide sequence analysis and homology search (BLAST). The results revealed that 7 of the 27 cDNA inserts were highly homologous to each other. One of the cDNA inserts, termed J6-2, was 690 bp in length and the encoding polypeptide was completely identical with the N-terminal half (amino acids 1–229) of MetAP2, a 478-amino acid glycoprotein (Fig. 1). We next examined whether the interaction between S100A4 and the polypeptide encoded by the J6-2 cDNA, hereafter called MetAP2-(1–229), takes place in vitro as well. For this purpose, a GST pull-down assay was employed. First, a construct prepared by subcloning theJ6-2 insert into pcDNA3/HA3 was subjected to thein vitro transcription-translation procedure. The resulting35S-labeled MetAP2-(1–229) was incubated with either GST or GST/mouse S100A4 fusion protein in the presence of calcium ion or EDTA. The fusion protein or GST was recovered by glutathione-Sepharose beads and the binding of MetAP2-(1–229) was assessed by SDS-PAGE followed by fluorography. As shown in Fig.2 A, MetAP2-(1–229) bound to neither GST nor GST/S100A4 fusion protein in the presence of EDTA, whereas it bound to the GST/S100A4 fusion protein but not to GST in the presence of the calcium ion. The same result was obtained using full-length mouse MetAP2, instead of MetAP2-(1–229), and human S100A4 (Fig. 2 B). Recombinant human MetAP2 was also shown to interact with human S100A4 (Fig. 2 C). On the other hand, human calmodulin, another member of the calcium-binding protein did not interact with mouse or human MetAP2 (Fig. 2, B andC). The above results prompted us to examine whether the interaction with MetAP2 is specific for S100A4 among S100 proteins. We chose human S100A1 and S100A6 on the basis of their same cytoplasmic localization and relatively high sequence homology with S100A4 and examined their interaction with MetAP2-(1–229) in a GST pull-down assay. As shown in Fig. 2 D, they were found to be able to interact with MetAP2-(1–229) in the presence of calcium ion. However, the intensity of the band differed in each other being in the order of S100A4 > S100A1 ≫ S100A6. To find out whether S100A4 interacts with MetAP2 in mammalian cells, 293 cells were transiently transfected with either the expression plasmid of FLAG-tagged S100A4 or that of HA-tagged MetAP2-(1–229) or both. The expression of the proteins in each transfectant was confirmed by immunoblot analysis (Fig. 3 A). The cell lysate was then subjected to immunoprecipitation with anti-S100A4 or anti-HA antibody followed by immunoblot analysis with anti-HA antibody. As shown in Fig. 3 B, the MetAP2-(1–229) was co-immunoprecipitated with S100A4 only from the cell lysate of the transfectants expressing both proteins. However, the amount of co-immunoprecipitated MetAP2-(1–229) was quite small, possibly because of a weak or transient interaction between S100A4 and MetAP2-(1–229) under our experimental conditions. We then treated the transfected cells with the membrane-permeable chemical cross-linking agent dithiobis(succinimidyl propionate) before cell lysis. The results shown in Fig. 3 B demonstrate that the amount of