Endogenous c-N-Ras Provides a Steady-state Anti-apoptotic Signal

doi:10.1074/jbc.M000250200

Endogenous c-N-Ras Provides a Steady-state Anti-apoptotic Signal*

Janice C. Wolfman‡ and
Alan Wolfman

From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Abstract

We report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser¹³⁶-pBad). Immortalized N-Ras knockout fibroblasts possess nearly undetectable levels of steady-state Ser¹³⁶-pBad. In contrast, wild-type control cells and the N-Ras knockout cells ectopically expressing c-N-Ras at control levels maintained easily detectable levels of Ser¹³⁶-pBad both at steady-state and following treatment with tumor necrosis factor α. Similar results were seen with Ser¹¹²-pBad. These differences did not arise from differences in total Bad protein levels. These data correlate with the observation that the N-Ras knockout cells exhibit a heightened susceptibility to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells at endogenous levels, compared with control cells, significantly rescues the apoptotically sensitive phenotype. Elevated expression of either c-Kirsten A-Ras or c-Kirsten B-Ras did not reverse the apoptotic sensitivity of the N-Ras knockout cells or result in increased levels of either phospho-Akt or phospho-Bad. Our results indicate that, at steady state, c-N-Ras possesses an isoform-specific, functional role in cell survival.

Footnotes

↵* This work was supported by American Heart Association Grant AHA96001110.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Cell Biology, NC10, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9752; Fax: 216-444-9404; E-mail: wolfmaj@ccf.org.
Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M000250200
Abbreviations:

Ha

Harvey

K(A) and K(B)

Kirsten A and B, respectively

PI

phosphatidylinositol

pAkt

serine 473-phosphorylated Akt

pBad

phosphorylated Bad

Ser¹³⁶-pBad

serine 136-phosphorylated Bad

Ser¹¹²-pBad

serine 112-phosphorylated Bad

ERK

extracellular signal-regulated kinase

MEK-1

MAP kinase-ERK1

MAP

mitogen-activated protein

MAPK

mitogen-activated protein kinase

HRP

horseradish peroxidase

MEF

mouse embryo fibroblast

PBS

phosphate-buffered saline

N−/−

N-Ras knockout cells

N+/−

N-Ras heterozygous cells

N+/+

control cells

N−/−wtN

N-Ras knockout cells ectopically expressing c-N-Ras at control cell levels

K−/−

K-Ras knockout cells

K+/+

control cells, MOPS, 3-(N-morpholino)propanesulfonic acid

CHAPS

3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid

TBS

Tris-buffered saline

PVDF

polyvinylidene difluoride

TUNEL

bromo-dUTP nick end labeling

ELISA

enzyme-linked immunosorbent assay

TNFα

tumor necrosis factor α
- Received January 12, 2000.
- Revision received March 20, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.