Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS

Science

Volumn 271, Issue 5250, 1996, Pages 810-812

  Joneson, Tom ^a   White, Michael A ^b   Wigler, Michael H ^b   Bar Sagi, Dafna ^a  

^a STONY BROOK UNIVERSITY   (United States)

^b Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GUANINE NUCLEOTIDE BINDING PROTEIN; MITOGEN ACTIVATED PROTEIN KINASE; MUTANT PROTEIN; RAS PROTEIN;

ARTICLE; CELL DIFFERENTIATION; CELL GROWTH; CONTROLLED STUDY; DNA SYNTHESIS; ENZYME ACTIVATION; FIBROBLAST; MEMBRANE RUFFLING; MITOGENESIS; PRIORITY JOURNAL; PROTEIN EXPRESSION; SIGNAL TRANSDUCTION;

EID: 0030052368     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5250.810     Document Type: Article

References (25)

1. A. F Chambers and A. B. Tuck, Crit. Rev. Oncog. 4, 95 (1993).
2. R. Davis, J. Biol. Chem. 268, 14553 (1993).
3. J Thorburn, M McMahon, A. Thorburn, ibid 269, 30580 (1994)
4. S Cowley et al., Cell 77, 841 (1994).
5. D. Bar-Sagi and J. R. Feramisco, Science 233, 1061 (1986)
6. V12S35 were cloned into the pDCR expression vector encoding the hemagglutinin (HA) non-apeptide epitope [I. Wilson et al , Cell 37, 767 (1984)], and RAF-CAAX was cloned into the pCDNA vector (Invitrogen). The 5XSRE-CAT expression plasmid was constructed as described [R. Graham and M. Gilman, Science 251, 189 (1991)].
7. V12S35 were cloned into the pDCR expression vector encoding the hemagglutinin (HA) non-apeptide epitope [I. Wilson et al , Cell 37, 767 (1984)], and RAF-CAAX was cloned into the pCDNA vector (Invitrogen). The 5XSRE-CAT expression plasmid was constructed as described [R. Graham and M. Gilman, Science 251, 189 (1991)].
8. H Sun, N K. Tonks, D. Bar-Sagi, Science 265, 285 (1994).
9. J. Blenis, Proc. Natl. Acad. Sci. USA 90, 5889 (1993)
10. For monitoring membrane ruffling, cells on cover slips were fixed in 3.7% formaldehyde in phosphate-buffered satine (PBS) for 1 hour at room temperature. The cells were then stained for 1 hour at room temperature in a humidified chamber with rhodamine-labeled phalloidin (0.1 μg/ml) dissolved in a solution of 90% PBS and 10% methanol. The cells were photographed with a Zeiss Axiophot fluorescence microscope
11. T. Joneson et al., unpublished data
12. M. A. White and M H. Wigler, unpublished data.
13. 32P]ATP with 0 2 mg/ml of myelin basic protein] Reaction products were analyzed by SDS-polyacrylamide gel electrophoresis and developed with the Fuji imaging system.
14. T. Joneson and D. Bar-Sagi, unpublished data.
15. A. Ridley et al., Cell 70, 401 (1992).
16. M. White et al , ibid. 80, 533 (1995).
17. For monitoring DNA synthesis, BrdU was added to the cell culture medium at a concentration of 10 μM 2 hours after injection After 30 hours, cells were fixed and permeabilized in acid alcohol (ethanol:water:acetic acid. 90:5:5) for 1 hour at -20°C. The cover slips were incubated with mouse antibody to BrdU and then with horseradish peroxidase-conjugated goat antibody to mouse immunoglobulin G (lgG). Immunocytochemical color development was done according to the manufacturer's instructions (Cell Proliferation Kit, Amersham). At least 100 injected cells were scored in each assay for quantitation.
18. S. J. Mansour et al., Science 265, 966 (1994).
19. R.-G. Qiu et al., Nature 374, 457 (1995).
20. H. Gille, A. D. Sharrocks, P E. Shaw, ibid. 358, 414 (1992); R. Marais, J Wynne, R. Treisman, Cell 73, 381 (1993).
21. H. Gille, A. D. Sharrocks, P E. Shaw, ibid. 358, 414 (1992); R. Marais, J Wynne, R. Treisman, Cell 73, 381 (1993).
22. O. Coso et al., Cell 81, 1137 (1995); A. Minden et al., ibid., p. 1147
23. O. Coso et al., Cell 81, 1137 (1995); A. Minden et al., ibid., p. 1147
24. For immunofluorescence, injected cells were fixed at the indicated times after injection in 3 7% formaldehyde in PBS for 1 hour at room temperature, then permeabilized with 0.1% Triton X-100 in PBS for 3 min at room temperature. The cover slips were incubated for 1 hour at 37°C with a mixture of either rat antibody to RAS (Y13-259) or mouse antibody to RAF (Transduction Laboratories) and rabbit antibody to CAT (5′→3′ Co ) in PBS containing bovine serum albumin (2 mg/ml) and then with a mixture of either fluorescein-conjugated goat antibody to rat lgG or goat antibody to mouse lgG and rhodamine-conjugated goat antibody to rabbit lgG (Cappel).
25. We thank S. Kaplan, L. Rodgers, and M Riggs fol technical assistance and L. VanAelst for the RAF-CAAX construct. Supported by NIH grant CA 55360 (D.B -S ) and by the American Cancer Society and National Cancer Institute (M.H W.). M H.W. is an American Cancer Society Research Professor.