Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells

Kiyoshi Ogura and Tadashi Tai1

Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113–8613, Japan

Received on March 8, 2001; revised on April 25, 2001; accepted on April 26, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We previously reported that GalCer expression factor 1 (GEF-1), a rat homologue of hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibition in COS-7 cells. In this study, we describe the characterization of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as dramatic morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significantly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is composed of four domains; a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/glutamine-rich (Q) domain. MDCK cells transfected with various GEF-1 deletion mutants were examined for morphology and for glycolipid expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were similar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-like cells, whereas those with the other deletion mutants showed no morphological changes, exhibiting typical epithelial-like cells. On the other hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells, but expressed sulfatide. The glycolipid expression paralleled CGT mRNA levels. Taking these results together, it is suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells.

Key words: galactosylceramide/sulfatide/MDCK cell/GEF-1


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Epithelial cell movement and changes in morphology are one of central issues in both development and regeneration of epithelial organs and are involved with pathological processes such as transformation of epithelia to carcinoma and metastasis (Balkovetz, 1998Go). Hepatocyte growth factor (HGF) is a mesenchymally derived growth factor with pleiotrophic effects on epithelia depending on culture conditions and plays a role in mesenchymal–epithelial interactions (Birchmeier and Birchmeier, 1993Go; Woolf et al., 1995Go). Madin-Darby canine kidney (MDCK) cells, which share many properties with polarized epithelia in vivo, are remarkably sensitive to HGF. In vitro models of HGF-treated MDCK cells have been proven to be useful for the study of epithelial cell movement and changes in morphology (Balkovetz, 1998Go). These phenomena have been shown to result in part from cytoskeletal reorganization, loss of intercellular junctions, and cell migration (Ridley et al., 1995Go; Janmey, 1998Go). Their mechanisms clearly depend on the activation of multiple intracellular molecular pathways (van der Geer et al., 1994Go).

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., 1998Go). 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, 1995Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Morphological changes of MDCK/GEF-1 cells
Significant differences in morphology were observed between MDCK/GEF-1 and parent MDCK cells transfected with pSV2–blasticidin S–resistant (bsr) (MDCK/bsr). As shown in Figure 1a and b, MDCK/GEF-1 cells exhibited a fibroblast-like stretched shape. These cells tended to be more stretched and piled up, and on day 7 were detached from flasks (Figure 1c). These detached cells were shown to be alive by means of the trypan blue exclusion test. In contrast, control MDCK/bsr cells exhibited a typical epithelial-like shape, forming tight monolayers with junctional complexes (Figure 1d,e). These cells reached flat confluent status and kept contact-inhibition (Figure 1f). Ratio of length and width in MDCK/GEF-1 cells on day 7 was 9.7 ± 3.2 (mean ± SD, N = 100), whereas the ratio in MDCK/bsr cells was 1.4 ± 0.3 (mean ± SD, N = 100). Thus MDCK/GEF-1 cells were shown to be about seven times more elongated than MDCK/bsr cells. These results suggested that the significant differences in morphology are observed between MDCK/GEF-1 and MDCK/bsr cells through all stages. No significant difference in thymidine incorporation was demonstrated between them, consistent with their similar cell growth rate (data not shown).



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Fig. 1. Morphological changes of MDCK/GEF-1 cells. Cells at 1, 3, and 7 days after plating are shown. (a, b, c) MDCK/GEF-1 cells; (d, e, f) control MDCK/bsr cells. (a, d) 1 day; (b, e) 3 days; (c, f) 7 days. The bar represents 100 µm.

 
Glycolipid expression in MDCK/GEF-1 cells
Thin-layer chromatography (TLC) analysis of glycolipids extracted from MDCK/GEF-1 cells revealed that GalCer-derived sulfatide expression was highly enhanced, whereas GalCer expression was similar compared to control cells (Figure 2, top). No significant changes were observed in the composition of other glycolipids. GalCer and sulfatide were confirmed using TLC immunostaining with monoclonal antibodies (MAbs) AMR20 (anti-GalCer) and AGB43 (anti-sulfatide), respectively (data not shown). The results suggested that the cells used may be MDCK strain I cells (Hansson et al., 1986Go), because sulfatide content was negligible in control MDCK cells. The expression of GalCer and sulfatide in MDCK/GEF-1 cells was also examined using an immunofluorescence technique with these MAbs. Sulfatide was detected in MDCK/GEF-1 cells, but not in MDCK/bsr (Figure 2, bottom). On the other hand, GalCer was not detected in MDCK/GEF-1 or MDCK/bsr cells (data not shown). Subsequently, CGT and cerebroside sulfotransferase (CST) activities were determined in the homogenate of MDCK/GEF-1 cells. MDCK/GEF-1 cells showed approximately twofold CGT enzyme activity compared to control MDCK/bsr cells (Figure 3, top). In contrast, CST activity was relatively high (14.6 ± 1.5 nmol/h/mg) in MDCK/GEF-1 and MDCK/bsr cells (data not shown). These results suggested that GEF-1 induced CGT activity but not CST activity in MDCK cells. Thus, we concluded that sulfatide expression was enhanced by the activation of CGT enzyme in MDCK/GEF-1 cells. We confirmed that the CGT mRNA, a single band, was detected at 3.2 kb in MDCK/GEF-1 cells, but not in control MDCK/bsr cells (Figure 3, bottom). The reason why the CGT mRNA was negligible in the control cells is unknown, probably due to the low sensitivity of the northern blot analysis. These results clearly indicated that GEF-1 is directly associated with CGT mRNA and induced CGT enzyme activity.




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Fig. 2. Expression of glycosphingolipids in MDCK/GEF-1 cells. Top, TLC analysis of glycosphingolipids from transfected MDCK/GEF-1 cells. Total lipid fraction extracted from the cells were developed on a borate-impregnated high-performance TLC plate in chloroform:methanol:5 M ammonium hydroxide 65:25:4 (v/v/v) and were visualized with orcinol stain. (1) MDCK/bsr; (2) MDCK/GEF-1; (3) standard sulfatide (2 µg); (4) standard GlcCer and GalCer (2 µg each). Lanes 1 and 2 correspond to 500 µg cell protein. Bottom, Immunofluorescence staining of MDCK/GEF-1 cells with anti-sulfatide antibody. Cells at 7 days after plating were stained. (a, b) MDCK/GEF-1 cells; (c, d) control MDCK/bsr cells. (a, c) Phase-contrast; (b, d) staining with MAb AGB43 (anti-sulfatide), followed by the incubation with FITC-conjugated goat F(ab')2 fragment to mouse IgG. The bar presents 100 µm.

 



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Fig. 3. Characterization of CGT in MDCK/GEF-1 cells. Top, CGT enzyme activity. Cell homogenates were incubated at 37°C for 30 min with C6 ceramide in the presence of UDP-14C-(U)-galactose (11.0 GBq/mmol) and 5 mM MnCl2 with 8 mM CHAPS. After the incubation, the lipids were extracted and separated by TLC. Radiolabeled spots were analyzed with a Bioimaging model BAS2000 analyzer. Results are expressed as the mean pmol/mg protein/h of triplicate determinations. (1) MDCK/bsr; (2) MDCK/GEF-1. Bottom, northern blot analysis of CGT mRNA. Ten micrograms of total RNAs per cells were hybridized with 32P-labeled CGT cDNA as a probe. Upper, CGT mRNA; lower, ethidium bromide–stained 18S rRNA. (1) MDCK/bsr; (2) MDCK/GEF-1.

 
Treatment of MDCK/GEF-1 cells with tyrosine kinase inhibitors and with microtubule inhibitors
We next examined whether tyrosine phosphorylation is involved in morphogenesis in MDCK/GEF-1 cells. A number of kinase inhibitors, including wortmannin, LY294002, tyrphostin 51, tyrphostin AG126, genistein, herbimycin A, rapamycin, and staurosporine, were added to the medium of cells and any morphological changes were assessed. Only tyrphostin 51, a potent inhibitor of growth factor receptor kinase, inhibited the morphological changes of MDCK/GEF-1 cells among a number of inhibitors tested (Figure 4b). Neither wortmannin (1 µM) nor LY294002, each of which is a potent inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), inhibited the morphological changes of MDCK/GEF-1 cells (data not shown). These results suggested that the tyrosine phosphorylation may be involved in morphological changes. Subsequently, microtubule inhibitors were added to the medium of cells and resulting morphological changes in MDCK/GEF-1 cells were observed. Treatment with either nocodazole (50 µM) or demecolcine (50 µM) reduced the length of the cells and the cells formed tight cell–cell junction (Figure 4c). MDCK/GEF-1 cells treated with these microtubule inhibitors were similar to parent MDCK cells in morphology. These findings suggested that alternations in the microtubule (tubulin) network resulted in morphological changes of MDCK/GEF-1 cells.



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Fig. 4. Effect of tyrosine phosphoryration inhibitors and microtubule polymerization inhibitors on MDCK/GEF-1 cells. Cells treated with tyrophostin 51 (10 µM) or with nocodazole (50 µM) at 2 days after plating and cultured for 1 day are shown. (a) Control MDCK/GEF-1 cells; no treatment. (b) MDCK/GEF-1 cells treated with tyrophostin 51. (c) MDCK/GEF-1 cells treated with nocodazole. The cells treated with demecolcine (50 µM) were similar to those with nocodazole. The bar represents 50 µm.

 
MDCK cells transfected with deletion mutants of GEF-1 cDNA
To determine which GEF-1 domains are involved in inducing morphological changes and in enhancing sulfatide expression in MDCK cells, we generated a number of stable MDCK transfectants with GEF-1 deletion mutants tagged with Flag. We confirmed that each transfectant cells expressed a specific deletion mutant protein by an immunoprecipitation/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis with an anti-Flag affinity gel (Figure 5, top left panel). These MDCK cells transfected with GEF-1 deletion mutants were then analyzed for morphology and for the glycolipid expression using an immunohistochemical technique. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with Z- and P-domains deletion mutant (MDCK/CQ) were similar to a wild-type GEF-1 (MDCK/ZPCQ) cells in shape, exhibiting fibroblast-like cells, whereas those with the other deletion mutants showed no morphological changes, exhibiting typical epithelial-like cells (Figure 5, top right panel, and Table I). On the other hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. The correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells in shape, but expressed sulfatide (Figure 5, top right panel, c and d). The elevation of CGT mRNA was detected in the glycolipid-positive transformant cells but not in the negative cells (data not shown). No cell growth suppression was observed in any of the MDCK transformant cells. These results suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells.






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Fig. 5. Characterization of GEF-1 deletion mutant proteins in various MDCK/GEF-1 transfectant cells. Top left panel, SDS–PAGE analysis. GEF-1 is composed of a zinc-finger motif (Z), a proline-rich region (P), a putative coiled-coil structure (C), and a proline/glutamine-rich region (Q). A wild-type GEF-1 and its nine deletion mutants were tagged with Flag. The deletion mutant cells labeled with 35S-methionine were lyzed and immunoprecipitated with an anti-Flag affinity gel. The immunoprecipitates were separated by SDS–PAGE under reduced conditions and were analyzed with a Bioimaging model BAS2000 analyzer. (1) ZPCQ (a wild type); (2) PCQ; (3) CQ; (4) Q; (5) Z; (6) P; (7) C; (8) ZP; (9) PC; (10) ZPC. Asterisks indicate corresponding deletion mutant proteins of GEF-1. Molecular mass standards are indicated in kilodaltons. Top right panel, Morphology and glycolipid expression. Cells at 3 days after plating are shown. (a, b) MDCK/ZPCQ cells. MDCK/PCQ and MDCK/CQ cells were similar to MDCK/ZPCQ; (c, d) MDCK/Q cells. (e, f) MDCK/Z cells. MDCK/P, MDCK/C, MDCK/ZP, MDCK/PC, and MDCK/ZPC cells were similar to MDCK/Z cells; (g, h) control MDCK/bsr cells. (a, c, e, g) Phase-contrast; (b, d, f, h) sulfatide expression. Sulfatide were detected using an immunofluorescence technique with a specific MAb AGB43. The bar represents 50 µm. Bottom left panel, Immunofluorescence staining of GEF-1 mutants. MDCK cells expressing the Flag-tagged GEF-1 deletion mutants were analyzed by anti-Flag antibody. (a, b) MDCK/ZPCQ; (c, d) MDCK/Q; (e, f) MDCK/Z; (g, h) control MDCK/bsr cells. The staining patterns of MDCK/PCQ and MDCK/CQ were similar to that of MDCK/ZPCQ. Those of MDCK/P, MDCK/C, MDCK/ZP, MDCK/PC, and MDCK/ZPC were similar to that of MDCK/Z. (a, c, e, g) Phase-contrast; (b, d, f, h) anti-Flag staining. The bar represents 50 µm. Bottom right panel, Tyrosine phosphorylation of GEF-1 mutants. MDCK/GEF-1 deletion mutants cells were lysed and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were separated by SDS–PAGE under reduced conditions and were immunoblotted with RC20 antibody specific for phospho-tyrosine. (1) MDCK/bsr; (2) MDCK/ZPCQ; (3) MDCK/PCQ; (4) MDCK/CQ; (5) MDCK/Q. Molecular mass standards are indicated in kDa. An arrowhead indicates 110-kDa GEF-1.

 

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Table I. Summary of MDCK cells transfected with various GEF-1 deletion mutants
 
Distribution of GEF-1 deletion mutant proteins in transfected MDCK cells
To elucidate the subcellular localization of wild-type GEF-1 and its deletion mutants in transfected MDCK cells, we performed indirect immunofluorescence staining of GEF-1. The wild-type GEF-1 was detected in perinuclear regions but not in cytoplasm, showing large vesicular structures (Figure 5, bottom left panel). Two deletion mutants including MDCK/PCQ and MDCK/CQ were similar to the wild-type transfectants. The Q-domain was also detected in the perinuclear regions, but showing small vesicular structures (Figure 5, bottom left panel). In contrast, the Z-domain and the other GEF-1 deletion mutants were detected diffusely in the cytoplasm (Figure 5, bottom left panel). Considering these results together, it was suggested that Q-domain would be essential for the localization of GEF-1 to the perinuclear regions.

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.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In the present study, we characterized the role of GEF-1 in MDCK cells. We have found that GEF-1 induces changes in the glycolipid expression and morphology (Figures 1 and 2), but not cell growth suppression. These results suggest that GEF-1 may be an adapter protein involving in signaling pathways to induce these phenomena and that MDCK cells may be more useful than COS-7 cells for the characterization of GEF-1 molecule in epithelial cells. In a previous study, the suppression of cell growth in COS-7 cells transfected with GEF-1 precluded us from a direct determination of their CGT mRNA level and characterization of GEF-1 molecule (Ogura et al., 1998Go). Thus, we have now demonstrated that both enzyme activity and mRNA level of CGT in MDCK/GEF-1 cells increase significantly compared to control cells (Figure 3). These findings clearly indicated that GEF-1 induces CGT mRNA, resulting in GalCer-derived sulfatide expression in MDCK cells.

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 cell–cell 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)Go 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, 2000Go). On the other hand, Asao et al. (1997)Go 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., 1998Go). 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, 1999Go). 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., 1998Go). Subsequently, Dupree et al. (1999)Go 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., 2000Go; Fujimoto et al., 2000Go).

Glycosphingolipids are believed to be integral components of plasma membrane microdomains, known as rafts and caveolae, that are rich in sphingolipids and cholesterol (Anderson, 1998Go; Brown and London, 1998Go). 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., 1998Go). 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., 1998Go). 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., 1999Go; Withers and Hakomori, 2000Go). 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)Go 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cells
Canine kidney epithelial cells, MDCK (Rindler et al., 1979Go) were purchased from RIKEN (Tsukuba, Japan). The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2.

Antibodies and reagents
Anti-GalCer MAb, AMR20 (IgM, {kappa}) and anti-sulfatide MAb, AGB43 (IgG3, {kappa}), both of which were generated and characterized in our laboratory (Kotani et al., 1994Go), 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 ({gamma} chain-specific) (diluted 1:200) were purchased from Cappel (Durham, NC). Horseradish peroxidase–conjugated goat anti-mouse Ig was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Horseradish peroxidase–conjugated 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., 1998Go). 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., 1996Go) 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)Go. 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
Met1–Leu233, Pro234–Pro390, Phe391–Phe562, and Pro563–Asp771 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.

SDS–PAGE 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 Tris–HCl (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 SDS–PAGE (5–20% 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 SDS–PAGE, 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 peroxidase–conjugated biotin and avidin DH complex of ABC kit. Tyrosine-phosphorylated proteins were visualized with the enhanced chemiluminescence western blotting detection system (Amersham Pharmacia Biotech).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (10178102) and Grant-in-Aid for Scientific Research (07458154 and 05780448) from the Ministry of Education, Science, Sports and Culture of Japan. The authors thank Dr. Popko (University of North Calorina) for providing CGT cDNA. They also thank Dr. K.O. Lloyd (Memorial Sloan-Kettering Cancer Center) for his critical reading of the manuscript.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
bsr, blasticidin S-resistant; CGT, UDP-galactose: ceramide galactosyltransferase; CST, cerebroside sulfotransferase; GalCer, galactosylceramide; GEF-1, GalCer expression factor-1; GlcCer, glucosylceramide; HGF, hepatocyte growth factor; Hrs, HGF-regulated tyrosine kinase substrate; MAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PBS, phosphate buffered saline; PI3-kinase, phosphatidylinositol 3-kinase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SH, Src homology; SSC, saline-sodium citrate; TLC, thin-layer chromatography.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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