Met is constitutively activated in those cells. The coordination of differentiation and proliferation is an essential feature in the successful development or replacement of tissues, but it is not clear how the decline of proliferation is related to the onset of differentiation. The expression of Met in lumen border cells in vivo and in vitro and the colocalization of Met and anti-P-Tyr immunofluorescence suggest that the Met receptor may be involved in differentiation toward lumen formation. Spatial organization might be dictated by the restricted distribution of the receptor (Fig. 3) (4). It is possible that the motogenic (scattering) activity of HGF/SF (28) is a fundamental part of this formation.
,
18. 19. 20.
21. 22. 23.
24. 25.
REFERENCES AND NOTES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10.
11. 12.
13. 14. 15. 16. 17.
C. S. Cooper etal., Nature 311, 29 (1984). M. Park et al., Cell 45, 895 (1986). S. Giordano et al., Oncogene 4, 1383 (1989). D. L. Faletto etal., ibid. 7, 1149 (1992). M. F. Di Renzo etal., ibid. 6, 1997 (1991). A. 1yer et al., Cell Growth Diff. 1, 87 (1990). D. P. Bottaro et al., Science 251, 802 (1991); L. Naldini etal., Oncogene 6, 501 (1991). G. K. Michalopoulos, FASEB J. 4, 176 (1990). T. Nakamura etal., Nature 342, 440 (1989); J. S. Rubin etal., Proc. Nat!. Acad. Sci. U.S.A. 88, 415 (1991); K. Tashiro et al., ibid. 87, 3200 (1990). E. Gerardi and M. Stoker, Cancer Cells 3, 227 (1991); L. Naldini etal., EMBO J. 10, 2876 (1991); K. M. Weidner et al., Proc. Nat!. Acad. Sci. U.S.A. 88, 7001 (1991). E. M. Rosen, J. Knesel, I. D. Goldberg, Cell Growth Diff. 2, 603 (1991). M. Stoker, E. Gherardi, M. Perryman, J. Gray, Nature 327, 239 (1987). R. Montesano, K. Matsumoto, L. Orci, Cell67, 901 (1991). I. Keydar et al., Eur. J. Cancer 15, 659 (1979). D. L. Faletto, unpublished results. M. Gonzatti-Haces et al., Proc. Nat!. Acad. Sci. U.S.A. 85, 21 (1988). For immunofluorescence assays, T47D cells treated with Bouin's fixative, breast cancer biopsies, and mouse embryos were paraffin-embedded, serial-sectioned (16), and blocked for 10 min with immunostaining kit blocking reagents (Biomeda, Foster City, CA). Primary antibody {C28, rabbit antibody to human Met [1:100 dilution in phosphate-buffered saline (PBS) (16)]; C200, rabbit antibody to the extracellular domain of the Met peptide, amino acids 643 to 663 [M. Park et al., Proc. Nat!. Acad. Sci. U.S.A. 84, 6379 (1987)]; SP260, rabbit antibody to mouse Met (1:100 dilution in PBS) (21); or 4G10, mouse monoclonal antibody to P-Tyr [D. K. Morrison et al., Cell 58, 649 (1989)]} was added and incubated for 2 hours at room temperature. Secondary antibody incubations (donkey antibody to rabbit immunoglobulin coupled to phycoerythrin diluted 1:50 in PBS and donkey antibody to mouse immunoglobulin coupled to fluorescein isothiocyanate diluted 1 :100 in PBS (Jackson lmmunoResearch Laboratories Westgrove, PA) were performed for 1 hour at room temperature. After an extensive washing, cells were fixed with gel mount (Biomeda). Fluorochrome-labeled cells were examined with a Zeiss CLSM with the following configuration: 25mW Ar and He-Ne lasers with 488, 514, and 543 maximum lines and control Indec (Sungate, Capitola, CA) software for image acquisition of x-y scan and z-series scan three-dimensional visualization. Fluorescent and Nomarski [J., Padawer, J. R. Microsc. Soc. 88, 305 (1968)] images were generated. Photographs were taken with a color
26. 27.
28.
29. 30.
g
video printer mavigraph (Sony) and UPC-5010a color print paper (Sony). When comparing the fluorescence intensity, we used identical parameters for each image (scanning line, laser light, contrast, and brightness) and assessed quantitation of the relative fluorescence by using Histogram, an Indec CLSM image processor option. I. Tsarfaty, unpublished results. Similar competition as in Fig. 5, A and B. We observed intense staining in duct-forming cells as compared to adjacent tissues in all samples similar to the staining in T47D cells (Fig. 2, C through E). H. Tajima, K. Matsumoto, T. Nakamura, FEBS Lett. 291, 229 (1991). M. Dean etal., Nature 318, 385 (1985); 1. Bieche etal., Lancet 339, 139 (1992). The quantitative determination of Met-specific immunofluorescence was performed as described in Fig. 2, C through E (17). M. Pratt et al., Int. J. Cancer 49, 323 (1991). Cells were grown on 16-chamber Labtek slides (Nunc, Roskild, Denmark) and treated with specific concentrations of HGF/SF (95% pure, Collaborative Research, Bedford, MA) for 24 hours. After two washes in PBS, cells were fixed for 10 min in cold methanol (-20°C) and washed extensively with PBS. Cells were also stained for 10 min with 0.1% methylene blue in PBS and were visualized and photographed with a Zeiss microscope. R. I. Freshney, Culture of Animal Cells: A Manual of Basic Technique (Liss, New York, ed. 2, 1987), pp. 57-84. K. Basler and E. Hafen, Science 243, 931 (1989); D. D. Bowtell, M. A. Simon, G. M. Rubin, Cell 56, 931 (1989). G. F. Vande Woude, Jpn. J. Cancer Res. 83, inside cover (1992). M. J. Bissell and G. Hall, in The Mammary Gland, M. Neville and C. Daniel, Eds. (Plenum, New York, 1987), pp. 97-146. C. H. Streuli, N. Bailey, M. J. Bissell, J. Cell Biol. 115, 1383 (1991).
31. Fixed cells were embedded in L.R. gold resin (Electron Microscopy Sciences) at -25°C, sectioned with a Nova Ultratome (LKB, Uppsala, Sweden), and picked up with Formvar-coated 200-mesh gold grids (Fort Washington, PA). The grids were washed three times in PBS for 10 min and incubated in 1% bovine serum albumin (BSA) in PBS for 2 hours and in C28 antibody (diluted 1: 50 in 1% BSA) at room temperature for 1 hour. Controls were incubated either in the presence of C28 competing peptide or in the absence of the primary antibody. The grids were washed again in PBS, incubated in RPMI 1640 medium for 20 min in 1% BSA, and reacted with goat antibody (to rabbit immunoglobulin) conjugated to gold (10-nm diameter; 1:10 diluted in 1% BSA; Amersham) at room temperature for 1 hour. The grids were finally washed in PBS and distilled water and stained with uranyl acetate and lead citrate. The sections were observed and photographed with an EM 410 electron microscope (Philips Eimdhoven, the Netherlands). 32. We thank D. Morrison for the gift of 4G10 anti-P-Tyr antibody; T. Papas and P. Pinto da Silva for the use of the confocal scanning-laser microscope and the Electron Microscopy Facility, respectively; S. Hughes, D. Kaplan, and 1. Daar for their critical review of this manuscript; and J. Hopkins and L. Summers for preparation of this manuscript. Supported in part by the National Cancer Institute, Department of Health and Human Services, under contract number NO1 -CO-741 01 with ABL. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. By acceptance of this report, the publisher or recipient acknowledges the right of the U.S. government and its agents and contractors to retain a nonexclusive, royalty-free license in and to any copyright covering the article. 7 April 1992; accepted 29 June 1992
Regulation of Protein Serine-Threonine Phosphatase Type-2A by Tyrosine Phosphorylation Jian Chen, Bruce L. Martin, David L. Brautigan* Extracellular signals that promote cell growth activate cascades of protein kinases. The kinases are dephosphorylated and deactivated by a single type-2A protein phosphatase. The catalytic subunit of type-2A protein phosphatase was phosphorylated by tyrosinespecific protein kinases. Phosphorylation was enhanced in the presence of the phosphatase inhibitor okadaic acid, consistent with an autodephosphorylation reaction. More than 90% of the activity of phosphatase 2A was lost when thioadenosine triphosphate was used to produce a thiophosphorylated protein resistant to autodephosphorylation. Phosphorylation in vitro occurred exclusively on Tyr307. Phosphorylation was catalyzed by p60v-src, p56'c
,
18. 19. 20.
21. 22. 23.
24. 25.
REFERENCES AND NOTES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10.
11. 12.
13. 14. 15. 16. 17.
C. S. Cooper etal., Nature 311, 29 (1984). M. Park et al., Cell 45, 895 (1986). S. Giordano et al., Oncogene 4, 1383 (1989). D. L. Faletto etal., ibid. 7, 1149 (1992). M. F. Di Renzo etal., ibid. 6, 1997 (1991). A. 1yer et al., Cell Growth Diff. 1, 87 (1990). D. P. Bottaro et al., Science 251, 802 (1991); L. Naldini etal., Oncogene 6, 501 (1991). G. K. Michalopoulos, FASEB J. 4, 176 (1990). T. Nakamura etal., Nature 342, 440 (1989); J. S. Rubin etal., Proc. Nat!. Acad. Sci. U.S.A. 88, 415 (1991); K. Tashiro et al., ibid. 87, 3200 (1990). E. Gerardi and M. Stoker, Cancer Cells 3, 227 (1991); L. Naldini etal., EMBO J. 10, 2876 (1991); K. M. Weidner et al., Proc. Nat!. Acad. Sci. U.S.A. 88, 7001 (1991). E. M. Rosen, J. Knesel, I. D. Goldberg, Cell Growth Diff. 2, 603 (1991). M. Stoker, E. Gherardi, M. Perryman, J. Gray, Nature 327, 239 (1987). R. Montesano, K. Matsumoto, L. Orci, Cell67, 901 (1991). I. Keydar et al., Eur. J. Cancer 15, 659 (1979). D. L. Faletto, unpublished results. M. Gonzatti-Haces et al., Proc. Nat!. Acad. Sci. U.S.A. 85, 21 (1988). For immunofluorescence assays, T47D cells treated with Bouin's fixative, breast cancer biopsies, and mouse embryos were paraffin-embedded, serial-sectioned (16), and blocked for 10 min with immunostaining kit blocking reagents (Biomeda, Foster City, CA). Primary antibody {C28, rabbit antibody to human Met [1:100 dilution in phosphate-buffered saline (PBS) (16)]; C200, rabbit antibody to the extracellular domain of the Met peptide, amino acids 643 to 663 [M. Park et al., Proc. Nat!. Acad. Sci. U.S.A. 84, 6379 (1987)]; SP260, rabbit antibody to mouse Met (1:100 dilution in PBS) (21); or 4G10, mouse monoclonal antibody to P-Tyr [D. K. Morrison et al., Cell 58, 649 (1989)]} was added and incubated for 2 hours at room temperature. Secondary antibody incubations (donkey antibody to rabbit immunoglobulin coupled to phycoerythrin diluted 1:50 in PBS and donkey antibody to mouse immunoglobulin coupled to fluorescein isothiocyanate diluted 1 :100 in PBS (Jackson lmmunoResearch Laboratories Westgrove, PA) were performed for 1 hour at room temperature. After an extensive washing, cells were fixed with gel mount (Biomeda). Fluorochrome-labeled cells were examined with a Zeiss CLSM with the following configuration: 25mW Ar and He-Ne lasers with 488, 514, and 543 maximum lines and control Indec (Sungate, Capitola, CA) software for image acquisition of x-y scan and z-series scan three-dimensional visualization. Fluorescent and Nomarski [J., Padawer, J. R. Microsc. Soc. 88, 305 (1968)] images were generated. Photographs were taken with a color
26. 27.
28.
29. 30.
g
video printer mavigraph (Sony) and UPC-5010a color print paper (Sony). When comparing the fluorescence intensity, we used identical parameters for each image (scanning line, laser light, contrast, and brightness) and assessed quantitation of the relative fluorescence by using Histogram, an Indec CLSM image processor option. I. Tsarfaty, unpublished results. Similar competition as in Fig. 5, A and B. We observed intense staining in duct-forming cells as compared to adjacent tissues in all samples similar to the staining in T47D cells (Fig. 2, C through E). H. Tajima, K. Matsumoto, T. Nakamura, FEBS Lett. 291, 229 (1991). M. Dean etal., Nature 318, 385 (1985); 1. Bieche etal., Lancet 339, 139 (1992). The quantitative determination of Met-specific immunofluorescence was performed as described in Fig. 2, C through E (17). M. Pratt et al., Int. J. Cancer 49, 323 (1991). Cells were grown on 16-chamber Labtek slides (Nunc, Roskild, Denmark) and treated with specific concentrations of HGF/SF (95% pure, Collaborative Research, Bedford, MA) for 24 hours. After two washes in PBS, cells were fixed for 10 min in cold methanol (-20°C) and washed extensively with PBS. Cells were also stained for 10 min with 0.1% methylene blue in PBS and were visualized and photographed with a Zeiss microscope. R. I. Freshney, Culture of Animal Cells: A Manual of Basic Technique (Liss, New York, ed. 2, 1987), pp. 57-84. K. Basler and E. Hafen, Science 243, 931 (1989); D. D. Bowtell, M. A. Simon, G. M. Rubin, Cell 56, 931 (1989). G. F. Vande Woude, Jpn. J. Cancer Res. 83, inside cover (1992). M. J. Bissell and G. Hall, in The Mammary Gland, M. Neville and C. Daniel, Eds. (Plenum, New York, 1987), pp. 97-146. C. H. Streuli, N. Bailey, M. J. Bissell, J. Cell Biol. 115, 1383 (1991).
31. Fixed cells were embedded in L.R. gold resin (Electron Microscopy Sciences) at -25°C, sectioned with a Nova Ultratome (LKB, Uppsala, Sweden), and picked up with Formvar-coated 200-mesh gold grids (Fort Washington, PA). The grids were washed three times in PBS for 10 min and incubated in 1% bovine serum albumin (BSA) in PBS for 2 hours and in C28 antibody (diluted 1: 50 in 1% BSA) at room temperature for 1 hour. Controls were incubated either in the presence of C28 competing peptide or in the absence of the primary antibody. The grids were washed again in PBS, incubated in RPMI 1640 medium for 20 min in 1% BSA, and reacted with goat antibody (to rabbit immunoglobulin) conjugated to gold (10-nm diameter; 1:10 diluted in 1% BSA; Amersham) at room temperature for 1 hour. The grids were finally washed in PBS and distilled water and stained with uranyl acetate and lead citrate. The sections were observed and photographed with an EM 410 electron microscope (Philips Eimdhoven, the Netherlands). 32. We thank D. Morrison for the gift of 4G10 anti-P-Tyr antibody; T. Papas and P. Pinto da Silva for the use of the confocal scanning-laser microscope and the Electron Microscopy Facility, respectively; S. Hughes, D. Kaplan, and 1. Daar for their critical review of this manuscript; and J. Hopkins and L. Summers for preparation of this manuscript. Supported in part by the National Cancer Institute, Department of Health and Human Services, under contract number NO1 -CO-741 01 with ABL. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. By acceptance of this report, the publisher or recipient acknowledges the right of the U.S. government and its agents and contractors to retain a nonexclusive, royalty-free license in and to any copyright covering the article. 7 April 1992; accepted 29 June 1992
Regulation of Protein Serine-Threonine Phosphatase Type-2A by Tyrosine Phosphorylation Jian Chen, Bruce L. Martin, David L. Brautigan* Extracellular signals that promote cell growth activate cascades of protein kinases. The kinases are dephosphorylated and deactivated by a single type-2A protein phosphatase. The catalytic subunit of type-2A protein phosphatase was phosphorylated by tyrosinespecific protein kinases. Phosphorylation was enhanced in the presence of the phosphatase inhibitor okadaic acid, consistent with an autodephosphorylation reaction. More than 90% of the activity of phosphatase 2A was lost when thioadenosine triphosphate was used to produce a thiophosphorylated protein resistant to autodephosphorylation. Phosphorylation in vitro occurred exclusively on Tyr307. Phosphorylation was catalyzed by p60v-src, p56'c
