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Requirement of Both Tyrosine Residues 1330 and 1337 in the C-Terminal Tail of the RON Receptor Tyrosine Kinase for Epithelial Cell Scattering and Migration
Biochemical and Biophysical Research Communications 267, 669 – 675 (2000) doi:10.1006/bbrc.1999.2011, available online at http://www.idealibrary.com on
Requirement of Both Tyrosine Residues 1330 and 1337 in the C-Terminal Tail of the RON Receptor Tyrosine Kinase for Epithelial Cell Scattering and Migration Z.-Q. Xiao, Y.-Q. Chen, and M.-H. Wang Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine and Denver Health Medical Center, Denver, Colorado 80204
Received December 14, 1999
RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON. © 2000 Academic Press
Receptor-type protein tyrosine kinases are a group of transmembrane protein that play a critical role in regulating cell growth, differentiation, and movement (1). The RON (recepteur d’origine nantais) receptor tyrosine kinase, also known as STK in mouse (2), is a 180 kD heterodimer composed of a 40 kD extracellular ␣ chain and a 150 kD transmembrane  chain with intrinsic protein tyrosine kinase activity (3). The ligand of RON was identified as macrophage stimulating protein (MSP) (4 – 6), also known as hepatocyte growth factor (HGF)-like protein (7). The roles of RON in cell transformation and tumor development are largely unknown, but under intensive investigation. RON is overexpressed in many primary breast carcinomas (8), and activation of RON causes invasive growth and motile activities of certain epithelial tumor cells (9, 10). Considering these facts, it was suggested that RON might
be involved in the process of epithelial tumor metastasis (11). RON belongs to the MET protooncogene family (12). Three members of this family (MET, RON and C-Sea) have been described (11) and characterized with regard to activities in cell morphology, and motility (11). Met is the receptor for HGF/scatter factor (SF) (13) and has well-described roles in cell transformation and tumor development (11). The C-Sea gene encodes a chicken cell surface receptor that is often overexpressed in transformed fibroblasts (14). All members of the MET family share many unique structural properties, including a putative proteolytic cleavage site, similar location of cysteine residues in their extracellular domain, and two conserved tyrosines in the C-terminal tail (12). These two tyrosine residues in the sequence of Y-hydrophobic-X-hydrophobic-(X) 3 -Y-hydrophobic-Nhydrophobic have been identified as a bidentate motif (15). In HGF-activated MET, this motif forms a multifunctional docking site, interacts with many SH-2 containing signaling proteins, such as Src and PI-3 kinase, and mediates different cellular functions including cell transformation (15). In RON, the function of the bidentate motif (Y 1330VQL-Y 1337MNL) has been related to growth and apoptosis of two-erythroleukemia cell lines (16). Whether this motif plays a role in RON-mediated epithelial cell migration and scattering has not been determined. The present studies address whether the interaction of the bidentate motif with PI-3 kinase is essential for RON-mediated cell migration and scattering. We found that both Y1330 and Y1337 are required for RON-mediated cell scattering and migration. Because double mutation leads to dissociation of RON with intracellular signaling proteins including PI-3 kinase, and a dominant inhibitory p85 of PI-3 kinase effectively blocks RON-mediated cell migration, we conclude that the interaction of PI-3 kinase with Y1330 and Y1337 in the bidentate motif is critical for
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FIG. 1. Expression of wtRON or RON mutants in kidney 293 cells. Cellular proteins (3 mg from each cell lines) were used for experiments. Rabbit IgG antibodies to RON peptide were used for both immunoprecipitation and Western blotting. 293 cells transfected with vector alone (293PC) service as negative control.
transducing motile signals in RON-activated epithelial cells. MATERIALS AND METHODS Cells and reagents. Human kidney 293 cells were from American Type Cell Collection (Rockville, MD). RE7 cells, a clone of MadinDarby canine kidney cells transfected with a human RON cDNA, were as described (4). Pure MSP was provided by Dr. E. J. Leonard (National Cancer Institute, NIH). Rabbit IgG antibodies against the synthetic C-terminal peptide of the RON -chain were as described (5). IgG antibodies to human p85 of PI-3 kinase or phospholipase C (PLC)-␥ were from Transduction Laboratories (Lexington, KY). Mouse IgG antibody to phosphotyrosine (clone 4G10) was from Upstate Biotechnology Inc (Lake Placid, NY). Enhanced chemiluminiscent (ECL) detection reagents were from Amersham (Arlington Heights, IL). Wortmannin was from Calbiochem (San Diego, CA). RPMI 1640 and DMEM were from Life Technologies (Gaithersburg, MD). Establishment of cell lines expressing wtRON or mutants. Mouse cDNAs encoding wild-type (wt) RON and three RON mutants, namely Y1330F, Y1337F, and Y1330F-1337F containing single or double substitution of tyrosine 1330 or 1337 with phenylalanine, were provided by Dr. T. Suda (University of Kumamoto, Japan) (16). Transfection of 293 cells with wtRON or mutant cDNAs was performed with transfection reagent DOTAP (Boehringer Mannheim) as described (4). Cells were selected with 800 g/ml of G418. Colonies were picked and expended into cell lines. Expression of wtRON or mutants was determined in Western blotting using rabbit IgG specific to mouse RON. Immunoprecipitation and Western blotting. Cells were lysed with 200 l lysis buffer (50 mM Tris buffer, pH 7.4, 0.5% Triton X-100, 0.5% NP-40, 150 mM NaCl, 2 mM EDTA, 100 mM vanadate, 25 g/ml leopeptin and aprotinin, and 50 g/ml soybean trypsin inhibitor). Proteins were mixed with rabbit IgG anti-RON or p85 of PI-3 kinase coupled with protein G-Sepharose beads. Samples were separated on an 8% polyacrylamide gel under reduced condition and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Western blotting was done with 4G10 anti-phosphotyrosine, followed by goat anti-mouse IgG conjugated with HRP, and developed with ECL reagents. In some experiments, the membrane was treated with SDS/2-mercaptoethanol erasure buffer and reprobed with other antibodies. Cell scatter assay. Cells (5 ⫻ 10 5/ml) were seeded in DMEM with 5% FBS at 0.5 ml/well in a 24-well tissue culture plate. After a 12 h
incubation, the medium was changed to DMEM with 1% FCS. MSP (5 nM) was added to each well. Cells without MSP were used as control. Cells were stained in Diff-Quick 24 h after incubation. Morphological changes were evaluated under the microscope and photographed. Cell migration assay. The assay was performed as previously described (4). Briefly, bottom wells of a multiwell chemotaxis chamber were filled with 30 l of DMEM containing different amounts of MSP in duplicate and then covered with a collagen IV-coated membrane. Upper wells were filled with 45 l of cell suspension (4 ⫻ 10 6 cells/ml in DMEM). The chamber was disassembled after a 5 h incubation at 37°C. The membranes were air-dried and stained with Diff-Quik. The migrated cells were counted in three random selected areas. Results were expressed as the percentage of input cells that migrated.
RESULTS Expression of wtRON or three mutants in 293 cells. Kidney 293 cells were chosen to express wtRON or RON mutants because of very low levels of endogenous RON (our unpublished data). More than 30 transfected cell lines were found expressing wtRON or individual RON mutants. Four cell lines, 293wtRON, RON1330F, RON1337F and RON1330/1337F, were chosen for functional analysis because the comparable levels of RON were observed. The results are shown in Fig. 1. MSP-induced phosphorylation of wtRON or mutants is shown in Fig. 2. No significant changes in levels of phosphorylation between wtRON and RON mutants were observed, suggesting that the mutation of Y1330, 1337, or both did not alter the phosphorylation status of RON. These results were confirmed also in other cells lines expressing wtRON or mutants (data not shown). We also tested protein kinase activities of the RON mutants. Reduction of enzymatic activities was not observed (data not shown). Effect of RON mutants on MSP-induced cell scattering. The results are shown in Fig. 3. Control 293PC cells did not respond to MSP. Cell grew as clusters. In contrast, 293wtRON cells displayed scatter-like activ-
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FIG. 2. Induction by MSP of phosphorylation of wtRON or mutants. Cells (3 ⫻ 10 6/ml) were stimulated with or without 5 nM of MSP at 37°C for 10 min. Immunoprecipitation was performed as described. Anti-phosphotyrosine (4G10) was used to detect phosphorelated RON (A). The membrane was treated with erasure buffer and reprobed with rabbit IgG to RON peptide to determine the amounts of RON (B).
ities after MSP stimulation. This effect was not observed in 293wtRON cells in the absence of MSP (date not shown). Interestingly, the scattering activity was also seen in RON1330F and RON1337F cells, indicating that substitution of Y1330 or Y1337 alone did not impair the morphological effect of RON. However, the scatter effect was abolished in RON1330/1337F cells. In these cells, clusters reappeared even in the presence of MSP. Similar results were also observed when additional clones were used (data not shown). Effect of RON mutants on MSP-induced cell migration. Because cell scattering is often accompanied by increased cell motility, we tested whether mutation of
Y1330 or Y1337 had any effect on RON-mediated cell migration. Results are shown in Fig. 4. 293wtRON cells migrated in a MSP concentration-dependent manner. Maximal cell migration was observed when 2.5 nM of MSP was added. RON1330F and RON1337F cells migrated also toward MSP. The number of migrated cells is comparable to those of 293wtRON cells, suggesting that mutation of Y1330 or Y1337 alone has no effect on RON-mediated cell motile activities. However, the migrating activities of RON1330/1337F cells were significantly reduced. The number of migrated cells is only approximately 50% of 293wtRON cells. These results were seen also in other cell clones ex-
FIG. 3. Effects of RON mutants on MSP-induced 293 cell scattering. Cells without MSP stimulation displaying unscattered morphology as shown in A are not presented. All cells shown here were stimulated with MSP. (A) 293PC cells as control; (B) 293wtRON cells; (C) RON1330F cells; (D) RON1337F; and (E) RON1330/1337F. One of three experiments with similar results. 671
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FIG. 4. Effect of RON mutants on MSP-induced 293 cell migration. The assay was performed as described under Materials and Methods. Cells migrated and adhered to the lower side of the membrane were counted under the microscope in three random selected areas. Results were expressed as means ⫾ SD. E, 293PC cells as control; F, 293wtRON cells; 䊐, RON1330F cells; ■, RON1337F cells; and Œ, RON1330/ 1337F cells. One of three experiments with similar results.
pressing RON or individual RON mutants (data not shown). These data, therefore, indicate that mutation of both Y1330 and Y1337 impairs the ability of RON to initiate cell movement. Interaction of p85 of PI-3 kinase with wtRON or RON mutants. The ability of RON or mutants to interact with p85 of PI-3 kinase was determined by coimmunoprecipitation. Results are shown in Fig. 5. The wtRON protein associated with p85 of PI-3 kinase following MSP stimulation. Mutant Y1330F or Y1337F
FIG. 5. MSP-induced association of p85 of PI-3 kinase with RON or mutants. (A) Detection of p85 of PI-3 kinase. Cells were stimulated with 5 nM MSP and precipitated with rabbit IgG to RON. Mouse IgG to p85 of PI-3 kinase was used in Western blotting. (B) Detection of RON or mutants. The membrane was stripped with erasure buffer and reprobed with rabbit IgG to RON peptide to determine the amounts of RON loaded in the membrane.
also interacted with p85 but half as much p85 was recruited to the mutant RON protein. In contrast, Y1330/1337F lost its ability to interact with p85 of PI-3 kinase. Experiments using additional cell clones expressing RON mutants confirmed these results (data not shown). These results demonstrate that both Y1330 and Y1337 are required for full association with p85 of PI-3 kinase. Effect of wortmannin on RON-mediated cell migration. To determine if PI-3 kinase plays a role in RON-mediated cell migration, a specific PI-3 kinase inhibitor, wortmannin, was included in cell migration assays. The results are shown in Fig. 6. Wortmannin inhibited the MSP-induced migration of 293wtRON cells in a dose-dependent manner. The 50% of inhibition were achieved when wortmannin was used at the concentration of 120 nM. Complete inhibition was observed when 500 nM Wortmannin was used (data not shown). Similar results were also obtained in RON1330F and RON1337F cells. In both cell lines, 50% of inhibition was achieved when wortmannin was used at 120 nM. The inhibitory effect of wortmannin on RON1330/1337F cells was insignificant. The effect of wortmannin on additional cell clones was also tested and the similar results were obtained (data not shown). These results confirmed our previous studies showing that Wortmannin inhibits MSP-induced keratinocyte migration, although sensitivities of individual cell lines to Wortmannin are different.
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FIG. 6. Effect of wortmannin on RON-mediated cell migration. Cells were pretreated with different amounts of wortmannin for 30 min. Cells were then added to the upper well of a multiwell chemotaxis chamber. Migrated cells adhered on the lower side of the membrane were counted. The data are shown from one experiment. Similar results were obtained from two similar experiments.
Effect of dominant-inhibitory (⌬) p85 of PI-3 kinase on RON-mediated cell migration. To exclude the possibility that wortmannin interferes with other signaling proteins which might be involved in cell migration, ⌬p85 of PI-3 kinase was used to block endogenous PI-3 kinase. Results are shown in Fig. 7. In 293wtRON cells tranafected with ⌬p85, RON-mediated cell migration was significantly reduced. The numbers of migrated cells was only at about 45% of original 293wtRON cells,
FIG. 7. Effect of ⌬p85 of PI-3 kinase on MSP-induced cell migration. 293wtRON cells were transiently transfected with 10 g of pcDNA3 containing ⌬p85 cDNA (F) or pcDNA3 vector alone (E) as a control. After a 24 h incubation, cells were used in migration assay. Results are from one experiment. Similar data were obtained from two other experiments.
indicating that inhibition of endogenous PI-3 kinase reduces RON-mediated cell migration. Similar results are confirmed with additional two cell lines expressing wtRON. Interestingly, ⌬p85 did not completely abrogate the effect of RON, suggesting that other signaling pathways may also involve in RON-mediated cell migration. DISCUSSION The central finding in this work is that both Y1330 and Y1337 are required for RON-mediated epithelial cell scattering and migration. Substitution of Y1330 or Y1337 alone is not sufficient to abrogate RONtransduced motile signals. Among activated signaling proteins, PI-3 kinase is the critical molecule which transduces the motile signals of RON. These findings support our notion that the bidentate motif is responsible for RON-mediated cell scattering and migration. The motile activities could be be important for RONmediated invasive growth of epithelial tumors in vivo (10). The bidentate motif formed by two adjacent tyrosines was originally identified in MET and subsequently found in all members of the MET family (12). Phosphorylation of Y1349 and Y1356 in MET is essential for cell transformation (15). Thus, it was predicted that Y1330 and Y1337 in the RON receptor would exert similar activities (15). However, functional studies have revealed that RON is different from other members of the MET family. RON does not have cell-
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transforming activity (9). Overexpression of RON only induces invasive-motile phenotype in epithelial tumor cells (10, 17). We hypothesized that in epithelial cells the Y1330-Y1337 docking site of RON is mainly responsible for transducing signals that regulate morphology and movement. The data presented here demonstrated that this is the case. Substitution of both tyrosine residues is sufficient to impair the ability of RON to initiate epithelial cell scattering and migration. Cell scattering and migration are two behaviors often observed in RON activated epithelial tumor cells (17, 18). However, the functional domains involved in these events, especially the bidentate motif, have not been studied. In MET, it has been shown that Y1356 (similar to Y1337 in RON) is critical for MET-mediated cell scattering (19). However, we found that introduction of single mutation at Y1330 or Y1337 has no effect on RON-mediated cell scattering and migration. Abrogation was achieved only when both tyrosines were mutated. These results suggest that unlike the MET receptor, in which mutation of Y1356 is sufficient to block cell scattering, the mutation of both Y1330 and Y1337 is essential for blocking RON-mediated motile signals. Our results differ also from those derived from studying the effect of RON mutant Y1330F or Y1337F on cell growth (16). In these studies, substitution Y1330 or Y1337 alone is sufficient to inhibit RONmediated cell proliferation (16). We believe that for RON to induce cell scattering or migration, phosphorylation of a tyrosine residue either at Y1330 or Y1337 alone is sufficient to activate down-stream signaling pathways. Moreover, because kinase activities are not affected by these mutations, the docking site formed by phosphorylation of either Y1330 or Y1337 is capable of associating with signaling proteins such as PI-3 kinase and initiating cell movement. The results presented in Figs. 4 and 5 support this conclusion. PI-3 kinase regulates cell migration and morphological changes (20) and has been implicated in the growth factor receptor-mediated tumor invasion of extracellular matrixes. We have previously shown that PI-3 kinase is an important enzyme involved in the RONmediated signaling events that lead to morphological change and migration (17). In keratinocytes, RON activation causes phosphorylation of p85 of PI-3 kinase, and induces the association of RON with PI-3 kinase (17). These effects are believed to account for the ability of RON to mediate cell migration and morphological change because wortmannin inhibits these activities (17). The present studies further extend the above findings and confirm the importance of PI-3 kinase in RON-mediated epithelial cell motility. We show that PI-3 kinase forms a complex with activated RON through the docking site created by phosphorylation of Y1330 or Y1337. Introduction of single mutation at Y1330 or Y1337 only partially affects the association.
Double mutation of Y1330 and Y1337 abolishes the interaction of RON with p85 of PI-3 kinase. Moreover, we show that expression of the dominant-inhibitory p85 of PI-3 kinase, which inhibits the endogenous PI-3 kinase (21), significantly blocks the RON-mediated migration of 293wtRON cells. These data, together with our previous findings, strongly suggest that PI-3 kinase is involved in transducing RON-mediated motile signals. Therefore, transduction of motile signlas by PI-3 kinase represents one of the common mechanisms among the members of the MET family. Recent studies have shown that RON is capable of activating multiple signaling pathways by interacting with different signal transducers through the bidentate motif (10, 16). These proteins include PLC-␥, PI-3 kinase, Sos, Src, MAPK/Erk2, and JNK/SAPK (22, 23). The functions of these signaling proteins in RONmediated cell scattering and migration are still unknown. Although our data suggest the importance of PI-3 kinase, they also imply the involvement of other mechanisms in the action of RON. As shown in Fig. 4, the substitution of both Y1330 and Y1337 only reduces RON-mediated cell migration by about 50%. A significant percentage of cells still migrate toward MSP. These results suggest that other functional domains in the RON protein are involved in transducing motile signals. Moreover, the inhibition of PI-3 kinase by wortmannin or by dominant inhibitory p85 only partially blocks RON-mediated cell migration (Figs. 6 and 7), indicating that signal transducers other than PI-3 kinase may also play a role. It will be of great interest in the future to determine these functional domains and signaling proteins. ACKNOWLEDGMENTS We thank Dr. T. Suda (University of Kumamoto, Japan) for cDNAs of RON mutants; Dr. E. J. Leonard (National Cancer Institute, Frederick, MD) for pure MSP, and Dr. M. Kasuga (Kobe University, Kobe, Japan) for cDNA of ⌬p85. The assistance of Ms. A. Casulo in manuscript preparation is greatly appreciated. This work was supported in part by a United States National Institutes of Health Grant RO1 AI43516.
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7. Han, S., Stuart, L. A., and Degen, S. J. F. (1991) Biochemistry 30, 9768 –9780. 8. Maggiora, P., Marchio, S., Stella, M. C., Giai, M., Belfiore, A., De Bortoli, M., Di Renzo, M. F., Costantino, A., Sismondi, P., and Comoglio, P. M. (1998) Oncogene 16, 2927–2933. 9. Santoro, M. M., Collesi, C., Grisendi, S., Gaudino, G., and Comoglio, P. M. (1996) Mol. Cell. Biol. 16, 7072–7083. 10. Collesi, C., Santoro, M. M., Gaudino, G., and Comoglio, P. M. (1996) Mol. Cell. Biol. 16, 5518 –5526. 11. Comoglio, P. M., and Boccaccio, C. (1996) Genes Cells 1, 347–354. 12. Rubin, J. S., Bottaro, D. P., and Aaronson, S. A. (1993) Biochim. Biophys. Acta 1155, 357–367. 13. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A, M., Kmiecik, T. E., Vande Woude, G. F., and Aaronson, S. A. (1993) Science 251, 802– 804. 14. Huff, J. L., Jelinek, M. A., Borgman, C. A., Lancing, T. L., and Parsons, J. T. (1993) Proc. Natl. Acad. Sci. USA 90, 6140 – 6144. 15. Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Zonca, P. D., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994) Cell 77, 261–271.
16. Iwama, A., Yamaguchi, N., and Suda, T. (1996) EMBO J. 15, 5866 –5875. 17. Wang, M.-H., Dlugosz, A. A., Sun, Y., Suda, T., Skeel, A., and Leonard, E. J. (1996) Exp. Cell Res. 226, 29 – 46. 18. Medico, E., Mongiovi, A. M., Huff, J. L., Jelinek, M. A., Follenzi, A., Gaodino, G., Parsons, J. T., and Comoglio, P. M. (1996) Mol. Biol. Cell. 7, 495–504. 19. Royal, I., and Park, M. (1995) J. Biol. Chem. 270, 27780 –2787. 20. Duronio, V., Scheid, M. P., and Ettinger, S. (1998) Cell Signal. 10, 233–239. 21. Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamura, Y., Sakaue, H., Ando, A., Chavanieu, A., Calas, B., and Grigorescu, F. (1994) EMBO J. 13, 2313–2321. 22. Li, B. Q., Wang, M.-H., Kung, F. H., Ronsin, C., Breathnach, R., Leonard, E. J., and Kamata, T. (1995) Biochem. Biophys. Res. Commun. 216, 110 –118. 23. Santoro, M. M., Penengo, L., Minetto, M., Orecchia, S., Cilli, M., and Gaodino, G. (1998) Oncogene 17, 741–749. 24. Pang, L., Sawada, T., Decker S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585–13588.
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Requirement of Both Tyrosine Residues 1330 and 1337 in the C-Terminal Tail of the RON Receptor Tyrosine Kinase for Epithelial Cell Scattering and Migration Z.-Q. Xiao, Y.-Q. Chen, and M.-H. Wang Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine and Denver Health Medical Center, Denver, Colorado 80204
Received December 14, 1999
RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON. © 2000 Academic Press
Receptor-type protein tyrosine kinases are a group of transmembrane protein that play a critical role in regulating cell growth, differentiation, and movement (1). The RON (recepteur d’origine nantais) receptor tyrosine kinase, also known as STK in mouse (2), is a 180 kD heterodimer composed of a 40 kD extracellular ␣ chain and a 150 kD transmembrane  chain with intrinsic protein tyrosine kinase activity (3). The ligand of RON was identified as macrophage stimulating protein (MSP) (4 – 6), also known as hepatocyte growth factor (HGF)-like protein (7). The roles of RON in cell transformation and tumor development are largely unknown, but under intensive investigation. RON is overexpressed in many primary breast carcinomas (8), and activation of RON causes invasive growth and motile activities of certain epithelial tumor cells (9, 10). Considering these facts, it was suggested that RON might
be involved in the process of epithelial tumor metastasis (11). RON belongs to the MET protooncogene family (12). Three members of this family (MET, RON and C-Sea) have been described (11) and characterized with regard to activities in cell morphology, and motility (11). Met is the receptor for HGF/scatter factor (SF) (13) and has well-described roles in cell transformation and tumor development (11). The C-Sea gene encodes a chicken cell surface receptor that is often overexpressed in transformed fibroblasts (14). All members of the MET family share many unique structural properties, including a putative proteolytic cleavage site, similar location of cysteine residues in their extracellular domain, and two conserved tyrosines in the C-terminal tail (12). These two tyrosine residues in the sequence of Y-hydrophobic-X-hydrophobic-(X) 3 -Y-hydrophobic-Nhydrophobic have been identified as a bidentate motif (15). In HGF-activated MET, this motif forms a multifunctional docking site, interacts with many SH-2 containing signaling proteins, such as Src and PI-3 kinase, and mediates different cellular functions including cell transformation (15). In RON, the function of the bidentate motif (Y 1330VQL-Y 1337MNL) has been related to growth and apoptosis of two-erythroleukemia cell lines (16). Whether this motif plays a role in RON-mediated epithelial cell migration and scattering has not been determined. The present studies address whether the interaction of the bidentate motif with PI-3 kinase is essential for RON-mediated cell migration and scattering. We found that both Y1330 and Y1337 are required for RON-mediated cell scattering and migration. Because double mutation leads to dissociation of RON with intracellular signaling proteins including PI-3 kinase, and a dominant inhibitory p85 of PI-3 kinase effectively blocks RON-mediated cell migration, we conclude that the interaction of PI-3 kinase with Y1330 and Y1337 in the bidentate motif is critical for
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FIG. 1. Expression of wtRON or RON mutants in kidney 293 cells. Cellular proteins (3 mg from each cell lines) were used for experiments. Rabbit IgG antibodies to RON peptide were used for both immunoprecipitation and Western blotting. 293 cells transfected with vector alone (293PC) service as negative control.
transducing motile signals in RON-activated epithelial cells. MATERIALS AND METHODS Cells and reagents. Human kidney 293 cells were from American Type Cell Collection (Rockville, MD). RE7 cells, a clone of MadinDarby canine kidney cells transfected with a human RON cDNA, were as described (4). Pure MSP was provided by Dr. E. J. Leonard (National Cancer Institute, NIH). Rabbit IgG antibodies against the synthetic C-terminal peptide of the RON -chain were as described (5). IgG antibodies to human p85 of PI-3 kinase or phospholipase C (PLC)-␥ were from Transduction Laboratories (Lexington, KY). Mouse IgG antibody to phosphotyrosine (clone 4G10) was from Upstate Biotechnology Inc (Lake Placid, NY). Enhanced chemiluminiscent (ECL) detection reagents were from Amersham (Arlington Heights, IL). Wortmannin was from Calbiochem (San Diego, CA). RPMI 1640 and DMEM were from Life Technologies (Gaithersburg, MD). Establishment of cell lines expressing wtRON or mutants. Mouse cDNAs encoding wild-type (wt) RON and three RON mutants, namely Y1330F, Y1337F, and Y1330F-1337F containing single or double substitution of tyrosine 1330 or 1337 with phenylalanine, were provided by Dr. T. Suda (University of Kumamoto, Japan) (16). Transfection of 293 cells with wtRON or mutant cDNAs was performed with transfection reagent DOTAP (Boehringer Mannheim) as described (4). Cells were selected with 800 g/ml of G418. Colonies were picked and expended into cell lines. Expression of wtRON or mutants was determined in Western blotting using rabbit IgG specific to mouse RON. Immunoprecipitation and Western blotting. Cells were lysed with 200 l lysis buffer (50 mM Tris buffer, pH 7.4, 0.5% Triton X-100, 0.5% NP-40, 150 mM NaCl, 2 mM EDTA, 100 mM vanadate, 25 g/ml leopeptin and aprotinin, and 50 g/ml soybean trypsin inhibitor). Proteins were mixed with rabbit IgG anti-RON or p85 of PI-3 kinase coupled with protein G-Sepharose beads. Samples were separated on an 8% polyacrylamide gel under reduced condition and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Western blotting was done with 4G10 anti-phosphotyrosine, followed by goat anti-mouse IgG conjugated with HRP, and developed with ECL reagents. In some experiments, the membrane was treated with SDS/2-mercaptoethanol erasure buffer and reprobed with other antibodies. Cell scatter assay. Cells (5 ⫻ 10 5/ml) were seeded in DMEM with 5% FBS at 0.5 ml/well in a 24-well tissue culture plate. After a 12 h
incubation, the medium was changed to DMEM with 1% FCS. MSP (5 nM) was added to each well. Cells without MSP were used as control. Cells were stained in Diff-Quick 24 h after incubation. Morphological changes were evaluated under the microscope and photographed. Cell migration assay. The assay was performed as previously described (4). Briefly, bottom wells of a multiwell chemotaxis chamber were filled with 30 l of DMEM containing different amounts of MSP in duplicate and then covered with a collagen IV-coated membrane. Upper wells were filled with 45 l of cell suspension (4 ⫻ 10 6 cells/ml in DMEM). The chamber was disassembled after a 5 h incubation at 37°C. The membranes were air-dried and stained with Diff-Quik. The migrated cells were counted in three random selected areas. Results were expressed as the percentage of input cells that migrated.
RESULTS Expression of wtRON or three mutants in 293 cells. Kidney 293 cells were chosen to express wtRON or RON mutants because of very low levels of endogenous RON (our unpublished data). More than 30 transfected cell lines were found expressing wtRON or individual RON mutants. Four cell lines, 293wtRON, RON1330F, RON1337F and RON1330/1337F, were chosen for functional analysis because the comparable levels of RON were observed. The results are shown in Fig. 1. MSP-induced phosphorylation of wtRON or mutants is shown in Fig. 2. No significant changes in levels of phosphorylation between wtRON and RON mutants were observed, suggesting that the mutation of Y1330, 1337, or both did not alter the phosphorylation status of RON. These results were confirmed also in other cells lines expressing wtRON or mutants (data not shown). We also tested protein kinase activities of the RON mutants. Reduction of enzymatic activities was not observed (data not shown). Effect of RON mutants on MSP-induced cell scattering. The results are shown in Fig. 3. Control 293PC cells did not respond to MSP. Cell grew as clusters. In contrast, 293wtRON cells displayed scatter-like activ-
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FIG. 2. Induction by MSP of phosphorylation of wtRON or mutants. Cells (3 ⫻ 10 6/ml) were stimulated with or without 5 nM of MSP at 37°C for 10 min. Immunoprecipitation was performed as described. Anti-phosphotyrosine (4G10) was used to detect phosphorelated RON (A). The membrane was treated with erasure buffer and reprobed with rabbit IgG to RON peptide to determine the amounts of RON (B).
ities after MSP stimulation. This effect was not observed in 293wtRON cells in the absence of MSP (date not shown). Interestingly, the scattering activity was also seen in RON1330F and RON1337F cells, indicating that substitution of Y1330 or Y1337 alone did not impair the morphological effect of RON. However, the scatter effect was abolished in RON1330/1337F cells. In these cells, clusters reappeared even in the presence of MSP. Similar results were also observed when additional clones were used (data not shown). Effect of RON mutants on MSP-induced cell migration. Because cell scattering is often accompanied by increased cell motility, we tested whether mutation of
Y1330 or Y1337 had any effect on RON-mediated cell migration. Results are shown in Fig. 4. 293wtRON cells migrated in a MSP concentration-dependent manner. Maximal cell migration was observed when 2.5 nM of MSP was added. RON1330F and RON1337F cells migrated also toward MSP. The number of migrated cells is comparable to those of 293wtRON cells, suggesting that mutation of Y1330 or Y1337 alone has no effect on RON-mediated cell motile activities. However, the migrating activities of RON1330/1337F cells were significantly reduced. The number of migrated cells is only approximately 50% of 293wtRON cells. These results were seen also in other cell clones ex-
FIG. 3. Effects of RON mutants on MSP-induced 293 cell scattering. Cells without MSP stimulation displaying unscattered morphology as shown in A are not presented. All cells shown here were stimulated with MSP. (A) 293PC cells as control; (B) 293wtRON cells; (C) RON1330F cells; (D) RON1337F; and (E) RON1330/1337F. One of three experiments with similar results. 671
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FIG. 4. Effect of RON mutants on MSP-induced 293 cell migration. The assay was performed as described under Materials and Methods. Cells migrated and adhered to the lower side of the membrane were counted under the microscope in three random selected areas. Results were expressed as means ⫾ SD. E, 293PC cells as control; F, 293wtRON cells; 䊐, RON1330F cells; ■, RON1337F cells; and Œ, RON1330/ 1337F cells. One of three experiments with similar results.
pressing RON or individual RON mutants (data not shown). These data, therefore, indicate that mutation of both Y1330 and Y1337 impairs the ability of RON to initiate cell movement. Interaction of p85 of PI-3 kinase with wtRON or RON mutants. The ability of RON or mutants to interact with p85 of PI-3 kinase was determined by coimmunoprecipitation. Results are shown in Fig. 5. The wtRON protein associated with p85 of PI-3 kinase following MSP stimulation. Mutant Y1330F or Y1337F
FIG. 5. MSP-induced association of p85 of PI-3 kinase with RON or mutants. (A) Detection of p85 of PI-3 kinase. Cells were stimulated with 5 nM MSP and precipitated with rabbit IgG to RON. Mouse IgG to p85 of PI-3 kinase was used in Western blotting. (B) Detection of RON or mutants. The membrane was stripped with erasure buffer and reprobed with rabbit IgG to RON peptide to determine the amounts of RON loaded in the membrane.
also interacted with p85 but half as much p85 was recruited to the mutant RON protein. In contrast, Y1330/1337F lost its ability to interact with p85 of PI-3 kinase. Experiments using additional cell clones expressing RON mutants confirmed these results (data not shown). These results demonstrate that both Y1330 and Y1337 are required for full association with p85 of PI-3 kinase. Effect of wortmannin on RON-mediated cell migration. To determine if PI-3 kinase plays a role in RON-mediated cell migration, a specific PI-3 kinase inhibitor, wortmannin, was included in cell migration assays. The results are shown in Fig. 6. Wortmannin inhibited the MSP-induced migration of 293wtRON cells in a dose-dependent manner. The 50% of inhibition were achieved when wortmannin was used at the concentration of 120 nM. Complete inhibition was observed when 500 nM Wortmannin was used (data not shown). Similar results were also obtained in RON1330F and RON1337F cells. In both cell lines, 50% of inhibition was achieved when wortmannin was used at 120 nM. The inhibitory effect of wortmannin on RON1330/1337F cells was insignificant. The effect of wortmannin on additional cell clones was also tested and the similar results were obtained (data not shown). These results confirmed our previous studies showing that Wortmannin inhibits MSP-induced keratinocyte migration, although sensitivities of individual cell lines to Wortmannin are different.
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FIG. 6. Effect of wortmannin on RON-mediated cell migration. Cells were pretreated with different amounts of wortmannin for 30 min. Cells were then added to the upper well of a multiwell chemotaxis chamber. Migrated cells adhered on the lower side of the membrane were counted. The data are shown from one experiment. Similar results were obtained from two similar experiments.
Effect of dominant-inhibitory (⌬) p85 of PI-3 kinase on RON-mediated cell migration. To exclude the possibility that wortmannin interferes with other signaling proteins which might be involved in cell migration, ⌬p85 of PI-3 kinase was used to block endogenous PI-3 kinase. Results are shown in Fig. 7. In 293wtRON cells tranafected with ⌬p85, RON-mediated cell migration was significantly reduced. The numbers of migrated cells was only at about 45% of original 293wtRON cells,
FIG. 7. Effect of ⌬p85 of PI-3 kinase on MSP-induced cell migration. 293wtRON cells were transiently transfected with 10 g of pcDNA3 containing ⌬p85 cDNA (F) or pcDNA3 vector alone (E) as a control. After a 24 h incubation, cells were used in migration assay. Results are from one experiment. Similar data were obtained from two other experiments.
indicating that inhibition of endogenous PI-3 kinase reduces RON-mediated cell migration. Similar results are confirmed with additional two cell lines expressing wtRON. Interestingly, ⌬p85 did not completely abrogate the effect of RON, suggesting that other signaling pathways may also involve in RON-mediated cell migration. DISCUSSION The central finding in this work is that both Y1330 and Y1337 are required for RON-mediated epithelial cell scattering and migration. Substitution of Y1330 or Y1337 alone is not sufficient to abrogate RONtransduced motile signals. Among activated signaling proteins, PI-3 kinase is the critical molecule which transduces the motile signals of RON. These findings support our notion that the bidentate motif is responsible for RON-mediated cell scattering and migration. The motile activities could be be important for RONmediated invasive growth of epithelial tumors in vivo (10). The bidentate motif formed by two adjacent tyrosines was originally identified in MET and subsequently found in all members of the MET family (12). Phosphorylation of Y1349 and Y1356 in MET is essential for cell transformation (15). Thus, it was predicted that Y1330 and Y1337 in the RON receptor would exert similar activities (15). However, functional studies have revealed that RON is different from other members of the MET family. RON does not have cell-
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transforming activity (9). Overexpression of RON only induces invasive-motile phenotype in epithelial tumor cells (10, 17). We hypothesized that in epithelial cells the Y1330-Y1337 docking site of RON is mainly responsible for transducing signals that regulate morphology and movement. The data presented here demonstrated that this is the case. Substitution of both tyrosine residues is sufficient to impair the ability of RON to initiate epithelial cell scattering and migration. Cell scattering and migration are two behaviors often observed in RON activated epithelial tumor cells (17, 18). However, the functional domains involved in these events, especially the bidentate motif, have not been studied. In MET, it has been shown that Y1356 (similar to Y1337 in RON) is critical for MET-mediated cell scattering (19). However, we found that introduction of single mutation at Y1330 or Y1337 has no effect on RON-mediated cell scattering and migration. Abrogation was achieved only when both tyrosines were mutated. These results suggest that unlike the MET receptor, in which mutation of Y1356 is sufficient to block cell scattering, the mutation of both Y1330 and Y1337 is essential for blocking RON-mediated motile signals. Our results differ also from those derived from studying the effect of RON mutant Y1330F or Y1337F on cell growth (16). In these studies, substitution Y1330 or Y1337 alone is sufficient to inhibit RONmediated cell proliferation (16). We believe that for RON to induce cell scattering or migration, phosphorylation of a tyrosine residue either at Y1330 or Y1337 alone is sufficient to activate down-stream signaling pathways. Moreover, because kinase activities are not affected by these mutations, the docking site formed by phosphorylation of either Y1330 or Y1337 is capable of associating with signaling proteins such as PI-3 kinase and initiating cell movement. The results presented in Figs. 4 and 5 support this conclusion. PI-3 kinase regulates cell migration and morphological changes (20) and has been implicated in the growth factor receptor-mediated tumor invasion of extracellular matrixes. We have previously shown that PI-3 kinase is an important enzyme involved in the RONmediated signaling events that lead to morphological change and migration (17). In keratinocytes, RON activation causes phosphorylation of p85 of PI-3 kinase, and induces the association of RON with PI-3 kinase (17). These effects are believed to account for the ability of RON to mediate cell migration and morphological change because wortmannin inhibits these activities (17). The present studies further extend the above findings and confirm the importance of PI-3 kinase in RON-mediated epithelial cell motility. We show that PI-3 kinase forms a complex with activated RON through the docking site created by phosphorylation of Y1330 or Y1337. Introduction of single mutation at Y1330 or Y1337 only partially affects the association.
Double mutation of Y1330 and Y1337 abolishes the interaction of RON with p85 of PI-3 kinase. Moreover, we show that expression of the dominant-inhibitory p85 of PI-3 kinase, which inhibits the endogenous PI-3 kinase (21), significantly blocks the RON-mediated migration of 293wtRON cells. These data, together with our previous findings, strongly suggest that PI-3 kinase is involved in transducing RON-mediated motile signals. Therefore, transduction of motile signlas by PI-3 kinase represents one of the common mechanisms among the members of the MET family. Recent studies have shown that RON is capable of activating multiple signaling pathways by interacting with different signal transducers through the bidentate motif (10, 16). These proteins include PLC-␥, PI-3 kinase, Sos, Src, MAPK/Erk2, and JNK/SAPK (22, 23). The functions of these signaling proteins in RONmediated cell scattering and migration are still unknown. Although our data suggest the importance of PI-3 kinase, they also imply the involvement of other mechanisms in the action of RON. As shown in Fig. 4, the substitution of both Y1330 and Y1337 only reduces RON-mediated cell migration by about 50%. A significant percentage of cells still migrate toward MSP. These results suggest that other functional domains in the RON protein are involved in transducing motile signals. Moreover, the inhibition of PI-3 kinase by wortmannin or by dominant inhibitory p85 only partially blocks RON-mediated cell migration (Figs. 6 and 7), indicating that signal transducers other than PI-3 kinase may also play a role. It will be of great interest in the future to determine these functional domains and signaling proteins. ACKNOWLEDGMENTS We thank Dr. T. Suda (University of Kumamoto, Japan) for cDNAs of RON mutants; Dr. E. J. Leonard (National Cancer Institute, Frederick, MD) for pure MSP, and Dr. M. Kasuga (Kobe University, Kobe, Japan) for cDNA of ⌬p85. The assistance of Ms. A. Casulo in manuscript preparation is greatly appreciated. This work was supported in part by a United States National Institutes of Health Grant RO1 AI43516.
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