CD44 Interaction with Tiam1 Promotes Rac1 Signaling and Hyaluronic Acid-mediated Breast Tumor Cell Migration
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.1829
ISSN1083-351X
AutoresLilly Bourguignon, Hongbo Zhu, Lijun Shao, You Wei Chen,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoIn this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH2-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393–738 of Tiam1) and an in vitrobinding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K d) of 0.2 nm, which is comparable with CD44 binding (K d = ∼0.13 nm) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression. In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH2-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393–738 of Tiam1) and an in vitrobinding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K d) of 0.2 nm, which is comparable with CD44 binding (K d = ∼0.13 nm) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression. hyaluronic acid pleckstrin homology PH domain located at the COOH-terminal region of the molecule coiled coil region extra region calmodulin-binding peptide green fluorescent protein glutathione S-transferase phosphate-buffered saline polymerase chain reaction guanosine 5′-3-O-(thio)triphosphate rhodamine fluorescein isothiocyanate The transmembrane glycoprotein CD44 isoforms are all major hyaluronic acid (HA)1 cell surface receptors that exist on many cell types, including macrophages, lymphocytes, fibroblasts, and epithelial cells (1.Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Google Scholar, 2.Picker L.J. Nakache M. Butcher E.C. J. Cell Biol. 1989; 109: 927-937Crossref PubMed Scopus (257) Google Scholar, 3.Bourguignon L.Y.W. 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Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (989) Google Scholar). Most often, the alternative splicing occurs between exons 5 and 15, leading to an insertion in tandem of one or more variant exons (v1–v10 (exon 6-exon 14) in human cells) within the membrane-proximal region of the extracellular domain (8.Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (989) Google Scholar). The variable primary amino acid sequence of different CD44 isoforms is further modified by extensive N- and O-glycosylations and glycosaminoglycan additions (9.Bennett K.L. Jackson D.G. Simon J.C. Tanczos E. Peach R. Modrell B. Stamenkivic I. Plowman G. Aruffo A. J. Cell Biol. 1995; 128: 687-698Crossref PubMed Scopus (369) Google Scholar, 10.Jackson D.G. Bell J.I. Dickinson R. Timans J. Shields J. Whittle N. J. 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Cell surface expression of CD44v isoforms changes profoundly during tumor metastasis, particularly during the progression of various carcinomas including breast carcinomas (13.Dall P. Heider K.-H. Sinn H-P Skroch-Angel P. Adolf G. Kaufmann M Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 14.Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 15.Kaufmann M. Meider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 16.Rodriguez C. Monges G. Rouanet P. Dutrillaux B. Lefrancois D. Theillet C. Int. J. Cancer. 1995; 64: 347-354Crossref PubMed Scopus (47) Google Scholar, 17.Kalish E. Iida N. Moffat E.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar). In fact, CD44v isoform expression has been used as an indicator of metastasis. It has been shown that interaction between the cytoskeletal protein, ankyrin, and the cytoplasmic domain of CD44 isoforms plays an important role in CD44 isoform-mediated oncogenic signaling (6.Bourguignon L.Y.W. Curr. Topics Membr. 1996; 43: 293-312Crossref Scopus (24) Google Scholar, 18.Bourguignon L.Y.W. Zhu D. Zhu H.B. Front. Biosci. 1998; 3: 637-649Crossref PubMed Scopus (108) Google Scholar, 19.Bourguignon L.Y.W. Iida N. Welsh C.F. Zhu D. Krongrad A. Pasquale D. J. Neuro-Oncol. 1995; 26: 201-208Crossref PubMed Scopus (54) Google Scholar). Specifically, the ankyrin-binding domain (e.g.NGGNGTVEDRKPSEL between amino acids 306 and 320 in the mouse CD44 (20.Lokeshwar V.B. Fregien N. Bourguignon L.Y.W. J. Cell Biol. 1994; 126: 1099-1109Crossref PubMed Scopus (201) Google Scholar) and NSGNGAVEDRKPSGL amino acids 304 and 318 in human CD44 (21.Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskelet. 1998; 39: 209-222Crossref PubMed Scopus (59) Google Scholar)) is required for the recruitment of Src kinase and the onset of tumor cell transformation (21.Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskelet. 1998; 39: 209-222Crossref PubMed Scopus (59) Google Scholar). Furthermore, HA binding to CD44 stimulates a concomitant activation of p185HER2-linked tyrosine kinase (linked to CD44s via a disulfide linkage) and results in a direct cross-talk between two different signaling pathways (e.g.proliferation versus motility/invasion) (22.Bourguignon L.Y.W. Zhu H. Chu A. Iida N. Zhang L. Hung H.C. J. Biol. Chem. 1997; 272: 27913-27918Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In tumor cells, the transmembrane linkage between CD44 isoform and the cytoskeleton promotes invasive and metastatic-specific tumor phenotypes (e.g. matrix degradation (matrix metalloproteinases) activities (23.Bourguignon L.Y.W. Gunja-Smith Z. Iida N. Zhu H.B. Young L.J.T. Muller W.J. Cardiff R.D. J. Cell. Physiol. 1998; 176: 206-215Crossref PubMed Scopus (245) Google Scholar, 24.Yu Q. Stamenkovic I. Genes Dev. 1999; 13: 35-48Crossref PubMed Scopus (608) Google Scholar), “invadopodia” formation (membrane projections), tumor cell invasion, and migration) (23.Bourguignon L.Y.W. Gunja-Smith Z. Iida N. Zhu H.B. Young L.J.T. Muller W.J. Cardiff R.D. J. Cell. Physiol. 1998; 176: 206-215Crossref PubMed Scopus (245) Google Scholar). These findings strongly suggest that the interaction between CD44 isoform and the cytoskeleton plays a pivotal role in the onset of oncogenesis and tumor progression. The Rho family proteins (e.g. Rho, Rac, and Cdc42) are members of the Ras superfamily of GTP-binding proteins structurally related to but functionally distinct from Ras itself (25.Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar, 26.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). They are associated with changes in the membrane-linked cytoskeleton (26.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). For example, activation of RhoA, Rac1, and Cdc42 have been shown to produce specific structural changes in the plasma membrane-cytoskeleton reorganization leading to membrane ruffling, lamellipodia, filopodia, and stress fiber formation (26.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). The coordinated activation of these GTPases is considered to be a possible mechanism underlying cell motility, an obvious prerequisite for metastasis (27.Dickson R.B. Lippman M.E. Mendelsohn J. Howlwy P.M. Israel M.A. Liotta L.A. The Molecular Basis of Cancer. W. B. Saunders Company, Philadelphia, PA1995: 358-359Google Scholar, 28.Jiang W.G. Puntis M.C.A. Hallett M.B. Br. J. Surgery. 1994; 81: 1576-1590Crossref PubMed Scopus (127) Google Scholar, 29.Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar). In particular, Rac1 activation is known to initiate oncogenic signaling pathways that promote cell shape changes (33.Ridley A.J. Paterson C.L. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar, 34.Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar), influence actin cytoskeleton organization (33.Ridley A.J. Paterson C.L. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar, 34.Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar), and stimulate gene expression (35.Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N.G. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar, 36.Minden A. Lin A.N. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 37.Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar). The question of whether Rac1 activation is also involved in CD44v3-related cytoskeleton function that results in the metastatic phenotypes (e.g. tumor cell migration) of breast tumor cells remains to be answered. Tiam1 (T lymphoma invasion andmetastasis 1) has been identified as an oncogene because of its ability to activate Rho-like GTPases during malignant transformation (38.Habets G.G.M. Scholtes E.H.M. Zuydgeest D. van der Kammen R.A. Stam J.C. Berns A. Collard J.G. Cell. 1994; 77: 537-549Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 39.Habets G.G.M. van der Kammen R.A. Stam J.C. Michiels F. Collard J.G. Oncogene. 1995; 10: 1371-1376PubMed Google Scholar). Specifically, Tiam1 is capable of activating Rac1 in vitro as a guanine nucleotide exchange factor and inducing membrane cytoskeleton-mediated cell shape changes, cell adhesion, and cell motility (34.Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar, 40.Michiels F. Habets G.G.M. Stan J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (509) Google Scholar, 41.Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 42.Van Leeuwen F.N. van der Kammen R.A. Habets G.G.M. Collard J.G. Oncogene. 1995; 11: 2215-2221PubMed Google Scholar). It also acts as a Rac-specific guanine nucleotide exchange factor in vivo and induces an invasive phenotypes in lymphoma cells (40.Michiels F. Habets G.G.M. Stan J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (509) Google Scholar). These findings have prompted several research groups to investigate the mechanisms involved in the regulation of Tiam1. For example, addition of certain serum-derived lipids (e.g. sphingosine-1-phosphate and lysophosphatidic acid) to T-lymphoma cells promotes Tiam1-mediated Rac1 and Cdc42 signaling and T-lymphoma cell invasion (43.Stam J.C. Michiels F. van der Kammen R.A. Moolenaar W.H. Collard J.G. EMBO J. 1998; 17: 4066-4074Crossref PubMed Scopus (203) Google Scholar). Tiam1 has also been found to be phosphorylated by protein kinase C in Swiss 3T3 fibroblasts stimulated by lysophosphatidic acid (44.Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and platelet-derived growth factor (45.Fleming I.N. Elliott C.M. Exton J.H. FEBS Lett. 1998; 429: 229-233Crossref PubMed Scopus (38) Google Scholar). Most recently, Exton and co-workers (46.Fleming I.N. Elliott C.M. Bruchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) demonstrate that phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II (but not protein kinase C) regulates Tiam1-catalyzed GDP/GTP exchange activity in vitro. These findings support the notion that posttranslational modifications of Tiam1 by certain serine/threonine kinase(s) during surface receptor-mediated activation may play an important role in Tiam1-Rac1 signaling. Tiam1 transcript has been detected in breast cancer cells (39.Habets G.G.M. van der Kammen R.A. Stam J.C. Michiels F. Collard J.G. Oncogene. 1995; 10: 1371-1376PubMed Google Scholar). However, it is not known at the present time whether there is any structural and functional relationship(s) between Tiam1-Rac1 signaling and CD44v3-mediated invasive and metastatic processes of breast cancer cells. In this paper, using a variety of biochemical, molecular biological, and immunocytochemical techniques, we have found that the cell adhesion molecule, CD44v3 isoform, which binds directly to HA, is closely associated with Tiam1 (in particular, the NH2-terminal pleckstrin homology (PHn), a putative coiled coil region (CC), and an additional adjacent region (Ex), designated as PHn-CC-Ex domain of Tiam1) in SP1 breast tumor cells. Most importantly, HA binding to CD44v3 isoform stimulates Tiam1-specific GDP/GTP exchange for Rho-like GTPases such as Rac1 and promotes cytoskeleton-mediated tumor cell migration. These findings suggest that a transmembrane interaction between CD44v3 and Tiam1 plays an important role in promoting oncogenic signaling and tumor cell-specific phenotypes required for HA-mediated breast tumor cell migration. Mouse breast tumor cells (e.g. SP1 cell line) (provided by Dr. Bruce Elliott, Department of Pathology, and Biochemistry, Queen's University, Kingston, ON, Canada) were used in this study. Specifically, SP1 cell line was derived from a spontaneous intraductal mammary adenocarcinoma that arose in a retired female CBA/J breeder in the Queen's University animal colony. These cells were capable of inducing lung metastases by sequential passage of SP1 cells into mammary gland (47.Elliott B.E. Maxwell L. Arnold M. Wei W.Z. Miller E.R. Cancer Res. 1988; 48: 7237-7245PubMed Google Scholar). These cells were cultured in RPMI 1640 medium supplemented with 5–7% fetal calf serum, folic acid (290 mg/liter), and sodium pyruvate (100 mg/liter). COS-7 cells were obtained from American Type Culture Collection and grown routinely in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin. For the preparation of polyclonal rabbit anti-Tiam1 antibody or rabbit anti-CD44v3 antibody, specific synthetic peptides (≈15–17 amino acids unique for the COOH-terminal sequence of Tiam1 or the CD44v3 sequence) were prepared by the Peptide Laboratories of Department of Biochemistry and Molecular Biology using an Advanced Chemtech automatic synthesizer (model ACT350). These Tiam1-related or CD44v3-related polypeptides were conjugated to polylysine and subsequently injected into rabbits to raise the antibodies, respectively. The anti-Tiam1-specific or anti-CD44v3-specific antibody was collected from each bleed and stored at 4 °C containing 0.1% azide. The anti-Tiam1 IgG or anti-CD44v3 IgG fraction was prepared by conventional DEAE-cellulose chromatography, respectively. Mouse monoclonal anti-HA (hemagglutinin epitope) antibody (clone 12 CA5) was purchased from Roche Molecular Biochemicals. Mouse monoclonal anti-green fluorescent protein (GFP) was purchased from PharMingen. Escherichia coli-derived GST-tagged Rac1 was kindly provided by Dr. Richard A. Cerione (Cornell University, Itheca, NY). SP1 cells suspended in PBS were surface labeled using the following biotinylation procedure. Briefly, cells (107 cells/ml) were incubated with sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce) (0.1 mg/ml) in labeling buffer (150 μm NaCl, 0.1 m HEPES, pH 8.0) for 30 min at room temperature. Cells were then washed with PBS to remove free biotin. Subsequently, the biotinylated cells were used for anti-CD44v3-mediated immunoprecipitation as described previously (23.Bourguignon L.Y.W. Gunja-Smith Z. Iida N. Zhu H.B. Young L.J.T. Muller W.J. Cardiff R.D. J. Cell. Physiol. 1998; 176: 206-215Crossref PubMed Scopus (245) Google Scholar). These biotinylated materials precipitated by anti-CD44v3 antibody were analyzed by SDS-polyacrylamide gel electrophoresis, transferred to the nitrocellulose filters, and incubated with ExtrAvidin-peroxidase (Sigma). After an addition of peroxidase substrate (Pierce), the blots were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions. SP1 cells were solubilized in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100 buffer and immunoprecipitated using rabbit anti-CD44v3 antibody or rabbit anti-Tiam1 antibody followed by goat anti-rabbit IgG, respectively. The immunoprecipitated material was solubilized in SDS sample buffer, electrophoresed, and blotted onto the nitrocellulose. After blocking nonspecific sites with 3% bovine serum albumin, the nitrocellulose filter was incubated with rabbit anti-Tiam1 antibody (5 μg/ml) or rabbit anti-CD44v3 antibody (5 μg/ml), respectively, for 1 h at room temperature followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution) at room temperature for 1 h. The blots were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions. In some experiments, SP1 cells or COS cells (e.g.untransfected or transfected by various Tiam1 cDNAs including the full-length mouse Tiam1cDNA (FL1591) or HA-tagged NH2-terminally truncated C1199 Tiam1cDNA or GFP-tagged PHn-CC-ExcDNA or C1199Taim1cDNA plus GFP-tagged PHn-CC-ExcDNA (as co-transfection) or vector only) were immunoblotted with mouse anti-HA antibody (5 μg/ml) or anti-GFP antibody (5 μg/ml), respectively, for 1 h at room temperature followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-mouse IgG (1:10,000 dilution) at room temperature for 1 h. The blots were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The procedure for preparing the fusion protein of the cytoplasmic domain of CD44 was the same as described previously (48.Bourguignon L.Y.W. Zhu H.B. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskelet. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar). Specifically, the cytoplasmic domain of human CD44 (CD44cyt) was cloned into pFLAG-AST using the PCR-based cloning strategy. Using human CD44 cDNA as template, one PCR primer pair (left, FLAG-EcoRI; right, FLAG-XbaI) was designed to amplify complete CD44 cytoplasmic domain. The amplified DNA fragments were one-step cloned into a pCR2.1 vector and sequenced. Then the DNA fragments were cut out by double digestion withEcoRI and XbaI and subcloned intoEcoRI/XbaI double-digested pFLAG-AST (Eastman Kodak Co., Rochester, NY) to generate FLAG-pCD44cyt construct. The nucleotide sequence of FLAG/CD44cyt junction was confirmed by sequencing. The recombinant plasmids were transformed to BL21-DE3 to produce FLAG-CD44cyt fusion protein. The FLAG-CD44cyt fusion protein was further purified by anti-FLAG M2 affinity gel column (Eastman Kodak Co.). The nucleotide sequence of primers used in this cloning protocol are: FLAG-EcoRI, 5′-GAGAATTCGAACAGTCGAAGAAGGTGTCTCTTAAGC-3′, and FLAG-XbaI, 5′-AGCTCTAGATTACACCCCAATCTTCAT-3′. Both the full-length mouse Tiam1cDNA (FL1591) and the NH2-terminally truncated Tiam1cDNA (C1199) were kindly provided by Dr. John G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Specifically, the full-length Taim1 (FL1591) cDNA was cloned into the eukaryotic expression vector, pMT2SM. The NH2-terminally truncated C1199 Tiam1 cDNA (carrying a HA epitope tag at the 3′ end) was cloned into the eukaryotic expression vector, pUTSV1 (Eurogentec, Belgium). The Tiam1 fragment, PHn-CC-Ex domain was cloned into calmodulin-binding peptide (CBP)-tagged vector (pCAL-n) (Stratagen) using the PCR-based cloning strategy. Using human Tiam1 cDNA as a template, PHn-CC-Ex domain was amplified by PCR with two specific primers (left, 5′-AACTCGAGATGAGTACCACCAACAGTGAG-3′, and right, 5′-AAAAAGCTTTCAGCCATCTGGAACAGTGTCATC-3′) linked with specific enzyme digestion site (XhoI or HindIII). PCR product digested with XhoI and HindIII was purified with QIAquick PCR Purification Kit (Qiagen). The PHn-CC-Ex domain cDNA fragment was cloned into pCAL-n vector digested withXhoI and HindIII. The inserted PHn-CC-Ex domain sequence was confirmed by nucleotide sequencing analyses. The recombinant plasmids were transformed to BL21-DE3 to produce CBP-tagged PHn-CC-Ex fusion protein. This fusion protein was purified from bacteria lysate by calmodulin affinity resin column (Sigma). The PHn-CC-Ex domain cDNA fragment was also cloned into pEGFPN1 vector (CLONTECH) digested with XhoI andHindIII to create GFP-tagged PHn-CC-Ex cDNA. The inserted PHn-CC-Ex domain sequence was confirmed by nucleotide sequencing analyses. This GFP-tagged PHn-CC-Ex domain cDNA was then used for transient expression in SP1 cells as described below. The molecular mass of the GFP-tagged PHn-CC-Ex is expressed as 68 kDa in SP1 or COS-7 cells by SDS-polyacrylamide gel electrophoresis and immunoblot analyses. To establish a transient expression system, SP1 cells (or COS-7 cells) were transfected with various plasmid DNAs (e.g. HA-tagged C1199 Tiam1cDNA, GFP-tagged PHn-CC-ExcDNA, or HA-tagged C1199Tiam1cDNA plus GFP-tagged PHn-CC-ExcDNA (as co-transfection) or vector alone) using electroporation methods according to those procedures described previously (74.Chu G. Hayakawa H. Berg P. Nucleic Acids Res. 1987; 15: 1311-1326Crossref PubMed Scopus (616) Google Scholar). Briefly, SP1 cells were plated at a density of 2 × 106 cells/100-mm dish and transfected with 25 μg/dish plasmid cDNA using electroporation at 230 V and 960 microfaraday with a Gene Pulser (Bio-Rad). Transfected cells were grown in the culture medium for at least 24–48 h. Various transfectants were then analyzed for their protein expression (e.g. Tiam1-related proteins) by immunoblot, GDP/GTP exchange reaction on Rac1, and tumor cell migration assays as described below. Aliquots (0.5–1 ng of protein) of purified FLAG-CD44cyt fusion protein bound to Anti-FLAG M2 antibody immunoaffinity beads were incubated in 0.5 ml of binding buffer (20 mm Tris-HCl (pH 7.4), 150 mmNaCl, 0.1% bovine serum albumin, and 0.05% Triton X-100) containing various concentrations (10–800 ng/ml) of 125I-labeled intact Tiam1 (purified from SP1 cells) (5000 cpm/ng protein) or125I-labeled recombinant Tiam1 fragment (CBP-tagged PHn-CC-Ex) at 4 °C for 4 h. Specifically, equilibrium binding conditions were determined by performing a time course (1–10 h) of125I-labeled Tiam1 (or CBP-tagged PHn-CC-Ex) binding to CD44 at 4 °C. The binding equilibrium was found to be established when the in vitro Tiam1 (or PHn-CC-Ex)-CD44 binding assay was conducted at 4 °C after 4 h. Following binding, the immunobeads were washed extensively in binding buffer, and the bead-bound radioactivity was counted. Nonspecific binding was determined using a 50–100-fold excess of unlabeled Tiam1 (or PHn-CC-Ex) in the presence of the same concentration of125I-labeled Tiam1 or 125I-labeled CBP-tagged PHn-CC-Ex. Nonspecific binding, which was approximately 20% of the total binding, was always subtracted from the total binding. Our binding data are highly reproducible. The values expressed in Fig. 5represent an average of triplicate determinations of three to five experiments with a standard deviation less than ± 5%. In some cases, 0.1 μg of surface biotinylated CD44v3 was incubated with various Tiam1-related proteins (e.g. purified intact Tiam1, HA-tagged C1199, CBP-PHn-CC-Ex, or HA/CBP-coated beads) in the presence and absence of 100-fold excess amount of CBP-PHn-CC-Ex at room temperature in the binding buffer (20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% bovine serum albumin, and 0.05% Triton X-100) for 1 h. After binding, biotinylated CD44v3 bound to the beads was analyzed by SDS-polyacrylamide gel electrophoresis, transferred to the nitrocellulose filters, and incubated with ExtrAvidin-peroxidase (Sigma). After an addition of peroxidase substrate (Pierce), the blots were developed using ECL chemiluminescence reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions. PurifiedE. coli-derived GST-tagged Rac1 (20pmol) was preloaded with GDP (30 μm) in 10 μl of buffer containing 25 mm Tris-HCl (pH 8.0), 1 mm dithiothreitol, 4.7 mm EDTA, 0.16 mm MgCl2, and 200 μg/ml bovine serum albumin at 37 °C for 7 min. To terminate preloading procedures, additional MgCl2 was then added to the solution (reaching a final concentration of 9.16 mm) as described previously (40.Michiels F. Habets G.G.M. Stan J.C. van der Kam
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