Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 1138613, Japan
Received on March 8, 2001; revised on April 25, 2001; accepted on April 26, 2001.
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Abstract |
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Key words: galactosylceramide/sulfatide/MDCK cell/GEF-1
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Introduction |
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We previously isolated a cDNA clone that induced galactosylceramide (GalCer) expression, morphological changes, and cell growth suppression in COS-7 cells from a rat brain cDNA library using a eukaryotic cell transient expression system (Ogura et al., 1998). The protein, designated GalCer expression factor 1 (GEF-1), was demonstrated to be a rat homologue of mouse HGF-regulated tyrosine kinase substrate (Hrs) (Komada and Kitamura, 1995
). In this study, we attempted to characterize the function of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK cells induced both glycolipid expression and morphological changes, but not cell growth suppression. The functional domains of GEF-1 molecule nesessary to elevate UDP-galactose:ceramide galactosyltransferase (CGT) mRNA level and to induce morphological changes were determined using MDCK cells transfected with various deletion mutants of GEF-1 cDNA. The results also would be useful for understanding the mechanisms by which HGF triggers cell motility and morphogenesis.
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Results |
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Western blot analysis of GEF-1 deletion mutant proteins
To determine which GEF-1 domains are phosphorylated with tyrosine in MDCK/GEF-1 cells, four deletion mutant proteins were analyzed by immunoprecipitation/western blot procedure. After Flag-tagged GEF-1 deletion mutant proteins were immunoprecipitated with a Flag-specific antibody, tyrosine phosphorylated-molecule was detected by RC20 antibody (Figure 5, bottom right panel). Among a number of GEF-1 deletion mutants tested, only a wild-type GEF-1 molecule was detected by the antibody, showing a 110-kDa band. In contrast, the other deletion mutant proteins were not detected clearly. These results suggested that the Z-domain of GEF-1 in MDCK/GEF-1 cells may be tyrosine-phosphorylated, whereas the other domains may not be. However, a possibility that the whole molecule of GEF-1 may be needed for the tyrosine phosphorylation at any domains still remains to be studied.
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Discussion |
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MDCK/GEF-1 cells exhibited a fibroblast-like stretched shape, tended to be piled up and were finally detached from flasks, but not dead (Figure 1). At present the precise mechanism of this phenomenon is unknown. GEF-1 signaling may always be switched on in MDCK/GEF-1 cells, inducing cellcell detachment. Drug inhibition tests in MDCK/GEF-1 cells revealed that (1) tyrphostin 51 inhibited the morphological changes (Figure 4b); (2) neither wortmannin nor LY294002 inhibited them; and (3) either nocodazole or demecolcine inhibited the morphological changes (Figure 4c). Domnina et al. (1985) described that the spreading and initial shape of epithelial cell lines are sensitive to treatment with microtubule depolymerizing drugs and that their cellular organization can be regulated by microtubule depolymerization. Considering these results together, it is suggested that GEF-1 protein may affect the morphology of MDCK cells through the polymerization of microtubules in the growth factor signal transduction system. In this study we characterized the effect of GEF-1 molecule on MDCK cells growing in monolayer culture. Further study will be needed to elucidate its roles in a collagen matrix culture.
A number of groups have recently characterized the functional domains of Hrs or GEF-1 using its deletion mutants. It was reported that Hrs localization to endosome requires the proline and glutamine-rich domain but not the FYVE finger (Hayakawa and Kitamura, 2000). On the other hand, Asao et al. (1997)
reported that the coiled-coil structure of Hrs is required for signaling from IL-2 to the nucleus in mouse T-cells. Our study clearly indicated that only the Q-domain is necessary for inducing the glycolipid expression, whereas the Q-domain together with the C-domain was essential for the role of GEF-1 to induce the morphological changes in MDCK cells. MDCK cells transfected with only the C-domain cDNA did not show any induction activity for either glycolipid expression or morphological changes (Figure 5, top right). In addition, our study revealed that the Q-domain is essential for the localization of GEF-1 to the perinuclear regions in MDCK cells (Figure 5, bottom left). The Q-domain has a number of consensus sequences for possible binding sites to the Src homology 2 (SH2) and SH3 domains of signal transduction molecules. In fact, two YXXM and YXXQ sequences for PI3-kinase and STAT3, respectively, exist in this domain (Ogura et al., 1998
). In this regard, it may be interesting to study more precisely possible binding sites by using site-directed mutagenesis. The identification of proteins associated with GEF-1, in particular with the C-Q and Q-domains, may be useful for elucidating their roles in the signaling pathways. A study along these lines is now being conducted.
Recently, knockout mice of the genes that associate with Hrs and GalCer expression have been reported. Hrs homozygous mutant embryos were shown to exhibit a defect in ventral folding morphogenesis and to die between E10.5 and E11.5 (Komada and Soriano, 1999). These results suggested that Hrs or GEF-1 may be required for development of several epithelial organs. On the other hand, mice with homozygous mutations in the CGT gene exhibited severe tremor and ataxia and die within 30 days after birth (Coetzee et al., 1998
). Subsequently, Dupree et al. (1999)
reported that myelin galactolipids are essential for the proper formation of axo-glial interactions and a disruption in these interactions results in profound abnormalities in the molecular organization of paranodal axolemma. Very recently, it was also reported that GalCer, sulfatide, or seminolipid is involved in the function of kidney and testis in mice (Tadano-Aritomi et al., 2000
; Fujimoto et al., 2000
).
Glycosphingolipids are believed to be integral components of plasma membrane microdomains, known as rafts and caveolae, that are rich in sphingolipids and cholesterol (Anderson, 1998; Brown and London, 1998
). These lipid domains assemble receptors and glycosylphosphatidylinositol-anchored proteins on their external surface and signaling molecules, Src-family kinases, G proteins, nitric oxide synthase, on their inner surface and mediate membrane trafficking and signal activity. The second type of glycosphingolipid domain consisting primarily of glycosphingolipids and signal transduction molecules has been proposed to couple cell adhesion interactions with signaling (Iwabuchi et al., 1998
). In this regard, it is interesting to study the relationship between glycolipid expression and morphological changes in MDCK cells. We described in a previous paper that there are two possibilities; one is that glycolipid expression may cause the morphological changes, the other is that glycolipid expression may occur concomitantly with morphological changes (Ogura et al., 1998
). The present study suggested that it is unlikely that glycolipid expression induced by GEF-1 results in the induction of morphological changes (Figure 5, upper right). Overexpression of CGT in MDCK cells, however, has shown sulfatide expression and some morphological changes (Ogura et al., unpublished observation). The overexpression of GEF-1 in MDCK cells may activate a number of specific signal transduction pathways, which may contribute to specific responses, including sulfatide expression and morphological changes. Further study will be needed to elucidate the relationship between them.
The present study indicated that GEF-1 induces CGT mRNA in MDCK cells. There are a number of reports in which signal transduction molecules, especially oncogenes such as src and ras, are involved in the regulation of glycosyl-transferases via the mitogen-activated protein kinase signaling pathways utilizing Ets-related transcription factors (Ko et al., 1999; Withers and Hakomori, 2000
). At present, however, there are no data on the transcription factors of CGT gene. Our study also indicated that GEF-1 is localized at the perinuclear region via the Q-domain in MDCK cells, although its fine distribution is not determined yet because of no useful markers available for the canine cells. Hayakawa and Kitamura (2000)
reported that Hrs is localized at the cytoplasmic surface of early endosomes in human HeLa cells by using an immunofluorescence technique. In this regard, further characterization is required to elucidate GEF-1 localization and to study the mechanism by which the Q-domain mediates interaction of GEF-1 with vesicle structures in MDCK cells.
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Materials and methods |
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Antibodies and reagents
Anti-GalCer MAb, AMR20 (IgM, ) and anti-sulfatide MAb, AGB43 (IgG3,
), both of which were generated and characterized in our laboratory (Kotani et al., 1994
), were purified from mouse ascites. Anti-Flag antibody M2 (diluted 1:100), M2-affinity gel and anti-phosphotyrosine recombinant antibody labeled with biotin, RC20 (diluted 1:1000), were obtained from Sigma-Aldrich Co. (St. Louis, MO) and Transduction Laboratories Inc. (Lexington, KY), respectively. FITC-conjugated goat F(ab')2 fragment to mouse IgM (µ chain-specific) (diluted 1:200) and to mouse IgG (
chain-specific) (diluted 1:200) were purchased from Cappel (Durham, NC). Horseradish peroxidaseconjugated goat anti-mouse Ig was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Horseradish peroxidaseconjugated biotin and avidin DH complex of ABC kit was purchased from Vector Laboratories (Burlingam, CA). A number of kinase inhibitors and microtubule inhibitors were obtained from Sigma-Aldrich Co. as follows; wortmannin, LY294002, tyrphostin 51, tyrphostin AG126, genistein, herbimycin A, rapamycin, staurosporine, demecolcine, and nocodazole. Glucosylceramide (GlcCer), GalCer (type 1), and sulfatide were purchased from Sigma-Aldrich Co.
Stable transfectants
Generation of stable transfectants of MDCK cells with GEF-1 cDNA was carried out as described previously (Ogura et al., 1998). Briefly, MDCK cells were co-transfected with 30 µg of pME-GEF-1 and 1 µg of pSV2bsr (Funakoshi, Japan) by electroporation. Bsr cells were selected and cloned by limiting dilution. A number of clones selected were similar in morphology, exhibiting fibroblast-like cells. A clone (MDCK/GEF-1) was characterized in this study.
Cell proliferation and thymidine incorporation assays
Cells were plated at 5 x 103 cells/well in 96-well plates. DNA synthesis was measured at 1 day after plating by adding 37kBq 3H-thymidine (Amersham Pharmacia Biotech). After incubation for 4 h, the cells were harvested with an automatic harvester, and 3H-thymidine incorporation was measured in a liquid scintillation counter.
Immunocytochemical analysis
Indirect immunofluorescence staining was performed as previously described (Kawashima et al., 1996) with minor modifications. Briefly, cells grown on LaboTec chamber slides were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) for 5min, followed by blocking with 5% bovine serum albumin in PBS for 1 h. They were incubated with an antibody for 1 h, and then washed with PBS. They were next incubated with FITC-conjugated goat F(ab')2 fragment to mouse IgM or IgG for 1 h. After washing, the stained cells were examined under an AXIOPHOT (Zeiss, Germany). Control experiments were performed with unrelated mouse Igs and gave no cellular stainings.
Analysis of glycosphingolipids
Transfected cells were havested by scraping, and washed with PBS three times. Total lipids were extracted from the cell (1 x 109) pellets by sonication with chloroform: methanol 2:1 (v/v, 200 ml), followed by treatment with mild alkaline. Glycosphingolipids were developed on a borate-impregnated high-performance TLC plate in a solvent system, chloroform:methanol:5 M ammonium hydroxide 65:25:4 (v/v/v) and were visualized with orcinol stain. Immunostaining on TLC plates were performed with peroxidase-conjugated secondary antibodies and were visualized with the enhanced chemiluminescence western blotting detection system (Amersham Pharmacia Biotech).
Assay for CGT activity
Cells grown for 3 days were harvested by scraping them off with a rubber policeman after rinsing with PBS, centrifuged, resuspended in PBS (50 x 106 cells/ml), and homogenized using a tight-fitting dounce homogenizer. Aliquots (50 µl) of the homogenates were incubated at 37°C for 30 min with short-chain ceramide (C6 ceramide, Matreya, Pleasant Gap, PA) in the presence of UDP-14C-(U)-galactose (11.0 GBq/mmol, NEN Lifescience Products Inc.) and 5 mM MnCl2 with 8 mM CHAPS. After the incubation the lipids were extracted and separated by TLC as described above, and were analyzed with a Bioimazing model BAS2000 analyzer (Fujix, Tokyo, Japan).
Assay for CST activity
CST activity was assayed according to the method of Kawano et al. (1989). Briefly, cells grown for 3 days were harvested, centrifuged, resuspended in Tris-buffered saline containing 0.1% Triton X-100 (50 x 106/ml), and homogenized as described above. Aliquots (50 µl) of the homogenates were incubated at 37°C for 1 h in the mixture of 25 mM MES buffer, pH 6.2, 100 mM 35S-3'-phosphoadenosine-5'-phosphosulfate (33KBq/mmol, NEN Lifescience Products Inc.), 2 mM ATP, 1.25 mM dithiothreitol, 5 mM NaF, 0.1 mM GalCer (type 1), 0.5% Triton X-100, 5 mM MnCl2, and 5 mM MgCl2. The lipids were analyzed with a similar procedure as described.
Northern blot analysis
A CGT clone was kindly supplied by B. Popko (University of North Carolina). The level of CGT mRNA in MDCK cells was analyzed by a routine procedure. 32P-labeled cDNA probe was prepared by random-primer labeling of a 2.4-kb EcoRI and NotI fragment of insert cDNA. The filter was hybridized with the 32P-labeled probe. After hybridization, the filter was subjected to washes of saline-sodium citrate (SSC; 1x SSC is 0.15 M NaCl and 0.05 M sodium citrate, pH 7.0) and 0.1% SDS for 15 min at room temperature, 0.1x SSC and 0.1% SDS for 2 h at 68°C.
Construction of the pcDNA3-Flag tagged GEF-1 deletion mutants
Met1Leu233, Pro234Pro390, Phe391Phe562, and Pro563Asp771 of GEF-1 were designated as a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/gultamine-rich (Q) domain, respectively. To construct the expression vector encoding a wild-type GEF-1 (ZPCQ) and its nine deletion mutants (PCQ, CQ, Q, Z, P, C, ZP, PC, and ZPC) tagged with Flag, we amplified Flag-epitoped mutant cDNA fragments by PCR method with the following each sense and antisense primer with pME-GEF-1 as a template. The Flag epitoped (DYLDDDDL) sense primer with EcoRI site were synthesized. Z sense primer: 5'-TTGAATTCATGGACTACAAGGACGACGATGACAAGATGGGGCGAGGCAGCGGCACC-3', P sense primer: 5'-TTGAATTCATGGACTACAAGGACGACGATGACAAGCCCCCAGAGTACCTGACCAGC-3', C sense primer: 5'-TTGAATTCATGGACTACAAGGACGACGATGACAAGTTTAGTGAGCAGTACCAGAAC-3', Q senseprimer: 5'-TTGAATTCATGGACTACAAGGACGACGATGACAAGCCCTTGCCTTATGCCCAGCTC-3'. The antisense primers with NotI site were synthesized. Z-antisense primer: 5'-ATAGTTTATGCGGCCGCTAAGTGGTAGAGGCAGCTTT-3', P-antisense primer: 5'ATAGTTTATGCGGCCGCTCAGGAAGTTATGGGCTGAGA-3', C-antisense primer: 5'-ATAGTTTATGCGGCCGCTAGGCACGCATCTGGACAGTCT-3', Q-antisense primer: 5'-ATAGTTTATGCGGCCGCTCAGTCGAAGGAGATGAGCTGGGT-3'. Amplified DNA fragments were digested with EcoRI and NotI. The resultant fragments were ligated to pcDNA3 (Invitrogen, The Netherlands) at the site of EcoRI and NotI. Each expression vector was sequenced to confirm the entire coding region sequence. Deletion mutant expression vectors were transfected into MDCK cells with a selection vector, pSV2-bsr, and bsr cells were selected and cloned by limiting dilution as described before.
SDSPAGE analysis of GEF-1 deletion mutant proteins
Transfectant cells labeled with 35S-methionine were lyzed with Mammalian Protein Extraction Reagent (1.0 ml) (Pierce, Rockford, IL) and centrifuged at 13,000 x g for 15 min. The supernatant, after preclearing with Sepharose CL-6B, was incubated for 16 h at 4°C under gentle agitation with anti-Flag antibody M2 affinity gel (30 µl). The gel was washed three times with 10 mM TrisHCl (pH 7.4) containing 150 mM NaCl, 0.1% Nonidet P-40, and 1 mM Na3VO4 (1.0 ml). The immunoprecipitated proteins were eluted with boiling SDS sample buffer (50 µl) and separated by SDSPAGE (520% polyacrylamide gel) under reducing conditions and were detected with a Bioimaging model BAS2000 analyzer.
Western blotting of GEF-1 deletion mutant proteins
Immunoprecipitated proteins were prepared as described above. After the SDSPAGE, the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Japan, Tokyo) and incubated with a recombinant RC20 antibody labeled with biotin for 1 h; these were followed by incubation for 1 h with horseradish peroxidaseconjugated biotin and avidin DH complex of ABC kit. Tyrosine-phosphorylated proteins were visualized with the enhanced chemiluminescence western blotting detection system (Amersham Pharmacia Biotech).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Asao, H., Sasaki, Y., Arita, T., Tanaka, N., Endo, K., Kasai, H., Takeshita, T., Endo, Y., Fujita, T., and Sugamura, K. (1997) Hrs is associated with STAM, a signal-transducing adaptor molecule: its suppressive effect on cytokine-induced cell growth. J. Biol. Chem., 272, 3278532791.
Balkovetz, D.F. (1998) Hepatocyte growth factor and Madin-Darby canine kidney cells: In vitro models of epithelial cell movement and morphogenesis. Microsc. Res. Tech., 43, 456463.[ISI][Medline]
Birchmeier, C. and Birchmeier, W. (1993) Molecular aspects of mesenchymal-epithelial interactions. Annu. Rev. Cell. Biol., 9, 511540.[ISI]
Brown, D.A. and London, E. (1998) Functions of lipid rafts in biological membranes. Annu. Rev. Cell. Dev. Biol., 14, 111136.[ISI][Medline]
Coetzee, T., Suzuki, K., and Popko, B. (1998) New perspectives on the function of myelin galactolipids. Trends Neurosci., 21, 126130.[ISI][Medline]
Domnina, L.V., Rovensky, J.A., Vasiliev, J.M., and Gelfand, I.M. (1985) Effect of microtubule-destroying drugs on the spreading and shape of cultured epithelial cells. J. Cell Sci., 74, 267282.[Abstract]
Dupree, J.L., Girault, J., and Popko, B. (1999) Axo-glial interactions regulate the localization of axonal paranodal proteins. J. Cell Biol., 147, 11451151.
Fujimoto, H., Tadano-Aritomo, K., Tokumasu, A., Ito, K., Hikita, T., Suzuki, K., and Ishizuka, I. (2000) Requirement of seminolipid in spermatogenesis revealed by UDP-galactose:ceramide galactosyltransferase-deficient mice. J. Biol. Chem., 275, 2262322626.
Hansson, G.C., Simons, K., and van Meer, G. (1986) Two strains of the Madin-Darby canine kidney (MDCK) cell line have two distinct glycosphingolipid compositions. EMBO J., 5, 483489.[Abstract]
Hayakawa, A. and Kitamura, N. (2000) Early endosomal localization of Hrs requires a sequence within the proline- and glutamine-rich region but not the FYVE finger. J. Biol. Chem., 275, 2963629642.
Iwabuchi, K., Handa, K., and Hakomori, S. (1998) Separation of glycosphingolipid signaling domain from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling. J. Biol. Chem., 273, 3376633773.
Janmey, P.A. (1998) The cytoskelton and cell signaling: component localization and mechanical coupling. Physiol. Rev., 78, 763781.
Kawano, M., Honke, K., Tachi, M., Gasa, S., and Makita, A. (1989) An assay method for ganglioside synthase using anion-exchange chromatography. Anal. Biochem., 182, 915.[ISI][Medline]
Kawashima, I., Nagata, I., and Tai, T. (1996) Immunocytochemical analysis of gangliosides in rat primary cerebellar cultures using specific monoclonal antibodies. Brain Res., 732, 7586.[ISI][Medline]
Ko, J.H., Miyoshi, E., Noda, K., Ekuni, A., Kang, R., Ikeda, Y., and Taniguchi, N. (1999) Regulation of the GnT-V promoter by transcription factor Ets-1 in various cancer cell lines. J. Biol. Chem., 274, 2294122948.
Komada, M. and Kitamura, N. (1995) Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-Kilodalton protein with a structurally conserved putative zinc finger domain. Mol. Cell. Biol., 15, 62136221.[Abstract]
Komada, M. and Soriano, P. (1999) Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. Genes Devel., 13, 14751485.
Kotani, M., Kawashima, I., Ozawa, H., Ogura, K., Ishizuka, I., Terashima, T., and Tai, T. (1994). Immunohistochemical localization of minor gangliosides in the rat central nervous system. Glycobiology, 4, 855865.[Abstract]
Ogura, K., Kohno, K. and Tai, T. (1998) Molecular cloning of a rat brain cDNA, with homology to a tyrosine kinase substrate, that induces galactosylceramide expression in COS-7 cells. J. Neurochem., 71, 18271836.[ISI][Medline]
Ridley, A.J., Comoglio, P. M., and Hall, A. (1995) Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol. Cell. Biol., 15, 11101122.[Abstract]
Rindler, M.J., Chuman, L.M., Shaffer, L., and Saier, M.H. (1979) Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK). J. Cell Biol., 81, 635648.[Abstract]
Tadano-Aritomi, K., Hikita, T., Fujimoto, H., Suzuki, K., Motegi, K., and Ishizuka, I. (2000) Kidney lipids in galactosylceramide synthase-deficient mice: absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids. J. Lipid Res., 41, 12371243.
van der Geer, P., Hunter, T., and Lindberg, R.A. (1994) Receptor protein-tyrosine kinases and their signal transduction pathways. Ann. Rev. Cell Biol., 10, 251337.
Withers, D.A. and Hakomori, S. (2000) Human (1, 3)-fucosyltransferase IV (FUTIV) gene expression is regulated by Elk-1 in the U937 cell line. J. Biol. Chem., 275, 4058840593.
Woolf, A.S., Kolatsi-Joannou, M., Hardman, P., Andermarcher, E., Moorby, C., Fine, L.G., Jat, P.S., Noble, M.D., and Gherardi, E. (1995) Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros. J. Cell Biol., 128, 171184.[Abstract]