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Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

Karassek S, Berghaus C, Schwarten M, Goemans CG, Ohse N, Kock G, Jockers K, Neumann S, Gottfried S, Herrmann C, Heumann R, Stoll R - J. Biol. Chem. (2010)

Bottom Line: We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner.Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis.NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiochemistry, Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, 44780 Bochum, Germany.

ABSTRACT
Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.

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Active c-Raf does not inhibit Rheb-enhanced apoptosis. A, Western blot showing that, in transfected HeLa cells, Rheb expression does not influence the activation level of endogenous c-Raf as indicated by no differences at the Ser-338 phosphorylation site of c-Raf. B and C, co-expression of a constitutively active c-Raf mutant significantly protects against UV light and TNFα-induced apoptosis but is not able to completely inhibit Rheb-enhanced apoptosis. D, representative Western blot illustrating the efficiency of ASK-1 knockdown. E and F, HeLa cells were transfected with scramble or human ASK-1 shRNA plus FLAG-Rheb-WT or FLAG-RhebΔCAAX and incubated for 96 h. ASK-1 knockdown led to a general decrease in cell death and inhibited Rheb-enhanced apoptosis after UV irradiation as well as TNFα stimulation, as shown by a lack of significant differences in cell death. All bars represent mean ± S.E. of at least six independent experiments. *, p < 0.05; ***, p < 0.001 (determined using ANOVA followed by a Bonferroni post hoc test).
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Figure 8: Active c-Raf does not inhibit Rheb-enhanced apoptosis. A, Western blot showing that, in transfected HeLa cells, Rheb expression does not influence the activation level of endogenous c-Raf as indicated by no differences at the Ser-338 phosphorylation site of c-Raf. B and C, co-expression of a constitutively active c-Raf mutant significantly protects against UV light and TNFα-induced apoptosis but is not able to completely inhibit Rheb-enhanced apoptosis. D, representative Western blot illustrating the efficiency of ASK-1 knockdown. E and F, HeLa cells were transfected with scramble or human ASK-1 shRNA plus FLAG-Rheb-WT or FLAG-RhebΔCAAX and incubated for 96 h. ASK-1 knockdown led to a general decrease in cell death and inhibited Rheb-enhanced apoptosis after UV irradiation as well as TNFα stimulation, as shown by a lack of significant differences in cell death. All bars represent mean ± S.E. of at least six independent experiments. *, p < 0.05; ***, p < 0.001 (determined using ANOVA followed by a Bonferroni post hoc test).

Mentions: Having established that Rheb binds specifically to c-Raf-RBD with low affinity in a Ras-like pattern, we tested whether this weak interaction might have functional implications in cells by measuring c-Raf activity using a phospho-specific c-Raf antibody against the activating phosphorylation site, Ser-338 (27). H-RasG12V increased phosphorylation of c-Raf (Fig. 8A). However, we were unable to detect major changes in the phosphorylation level of c-Raf in the presence of Rheb-WT, which is consistent with the notion that, because of the low binding affinity in the micromolar range, direct regulation of the c-Raf kinase might not be possible, despite the interaction specificity.


Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

Karassek S, Berghaus C, Schwarten M, Goemans CG, Ohse N, Kock G, Jockers K, Neumann S, Gottfried S, Herrmann C, Heumann R, Stoll R - J. Biol. Chem. (2010)

Active c-Raf does not inhibit Rheb-enhanced apoptosis. A, Western blot showing that, in transfected HeLa cells, Rheb expression does not influence the activation level of endogenous c-Raf as indicated by no differences at the Ser-338 phosphorylation site of c-Raf. B and C, co-expression of a constitutively active c-Raf mutant significantly protects against UV light and TNFα-induced apoptosis but is not able to completely inhibit Rheb-enhanced apoptosis. D, representative Western blot illustrating the efficiency of ASK-1 knockdown. E and F, HeLa cells were transfected with scramble or human ASK-1 shRNA plus FLAG-Rheb-WT or FLAG-RhebΔCAAX and incubated for 96 h. ASK-1 knockdown led to a general decrease in cell death and inhibited Rheb-enhanced apoptosis after UV irradiation as well as TNFα stimulation, as shown by a lack of significant differences in cell death. All bars represent mean ± S.E. of at least six independent experiments. *, p < 0.05; ***, p < 0.001 (determined using ANOVA followed by a Bonferroni post hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962498&req=5

Figure 8: Active c-Raf does not inhibit Rheb-enhanced apoptosis. A, Western blot showing that, in transfected HeLa cells, Rheb expression does not influence the activation level of endogenous c-Raf as indicated by no differences at the Ser-338 phosphorylation site of c-Raf. B and C, co-expression of a constitutively active c-Raf mutant significantly protects against UV light and TNFα-induced apoptosis but is not able to completely inhibit Rheb-enhanced apoptosis. D, representative Western blot illustrating the efficiency of ASK-1 knockdown. E and F, HeLa cells were transfected with scramble or human ASK-1 shRNA plus FLAG-Rheb-WT or FLAG-RhebΔCAAX and incubated for 96 h. ASK-1 knockdown led to a general decrease in cell death and inhibited Rheb-enhanced apoptosis after UV irradiation as well as TNFα stimulation, as shown by a lack of significant differences in cell death. All bars represent mean ± S.E. of at least six independent experiments. *, p < 0.05; ***, p < 0.001 (determined using ANOVA followed by a Bonferroni post hoc test).
Mentions: Having established that Rheb binds specifically to c-Raf-RBD with low affinity in a Ras-like pattern, we tested whether this weak interaction might have functional implications in cells by measuring c-Raf activity using a phospho-specific c-Raf antibody against the activating phosphorylation site, Ser-338 (27). H-RasG12V increased phosphorylation of c-Raf (Fig. 8A). However, we were unable to detect major changes in the phosphorylation level of c-Raf in the presence of Rheb-WT, which is consistent with the notion that, because of the low binding affinity in the micromolar range, direct regulation of the c-Raf kinase might not be possible, despite the interaction specificity.

Bottom Line: We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner.Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis.NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiochemistry, Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, 44780 Bochum, Germany.

ABSTRACT
Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.

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