title: R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.
abstract: The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.
authors.affiliation: La Jolla Cancer Research Foundation, California 92037, USA.
authors.forename: H G
authors.initials: HG
authors.lastname: Wang
authors.forename: J A
authors.initials: JA
authors.lastname: Millan
authors.forename: A D
authors.initials: AD
authors.lastname: Cox
authors.forename: C J
authors.initials: CJ
authors.lastname: Der
authors.forename: U R
authors.initials: UR
authors.lastname: Rapp
authors.forename: T
authors.initials: T
authors.lastname: Beck
authors.forename: H
authors.initials: H
authors.lastname: Zha
authors.forename: J C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002999:Clone Cells; D016895:Culture Media, Serum-Free; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006133:Growth Substances / Q000172:deficiency*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D019908:Proto-Oncogene Proteins c-raf; D013489:Suppression, Genetic; D014162:Transfection; D051028:bcl-2-Associated X Protein; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D016895:Culture Media, Serum-Free; D006133:Growth Substances; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C513254:Rras protein, mouse; D018631:ras Proteins