R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism

Hong Gang Wang, Juan A. Millan, Adrienne D. Cox, Channing J. Der, Ulf R. Rapp, Thomas Beck, Hongbin Zha, John C. Reed

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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.

Original languageEnglish (US)
Pages (from-to)1103-1114
Number of pages12
JournalJournal of Cell Biology
Volume129
Issue number4
DOIs
StatePublished - May 1995

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Intercellular Signaling Peptides and Proteins
Apoptosis
Cell Death
Interleukin-3
Proto-Oncogene Proteins c-raf
ras Proteins
Guanosine Triphosphate
Cell Survival
Plasmids
Sf9 Cells
NIH 3T3 Cells
Proteins
Monomeric GTP-Binding Proteins
Baculoviridae
GTP Phosphohydrolases
Valine
Glycine
Clone Cells
Serum

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Wang, Hong Gang ; Millan, Juan A. ; Cox, Adrienne D. ; Der, Channing J. ; Rapp, Ulf R. ; Beck, Thomas ; Zha, Hongbin ; Reed, John C. / R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. In: Journal of Cell Biology. 1995 ; Vol. 129, No. 4. pp. 1103-1114.
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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.",
author = "Wang, {Hong Gang} and Millan, {Juan A.} and Cox, {Adrienne D.} and Der, {Channing J.} and Rapp, {Ulf R.} and Thomas Beck and Hongbin Zha and Reed, {John C.}",
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Wang, HG, Millan, JA, Cox, AD, Der, CJ, Rapp, UR, Beck, T, Zha, H & Reed, JC 1995, 'R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism', Journal of Cell Biology, vol. 129, no. 4, pp. 1103-1114. https://doi.org/10.1083/jcb.129.4.1103

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. / Wang, Hong Gang; Millan, Juan A.; Cox, Adrienne D.; Der, Channing J.; Rapp, Ulf R.; Beck, Thomas; Zha, Hongbin; Reed, John C.

In: Journal of Cell Biology, Vol. 129, No. 4, 05.1995, p. 1103-1114.

Research output: Contribution to journalArticle

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T1 - R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism

AU - Wang, Hong Gang

AU - Millan, Juan A.

AU - Cox, Adrienne D.

AU - Der, Channing J.

AU - Rapp, Ulf R.

AU - Beck, Thomas

AU - Zha, Hongbin

AU - Reed, John C.

PY - 1995/5

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N2 - 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.

AB - 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.

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