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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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Steady state heteronuclear NOE for the backbone amides of rRheb in its GDP- (A) and Gpp(NH)p-bound (B) states. Residues for which no results are shown correspond to either prolines or residues for which relaxation data could not be extracted. The switch I region extends from residue Asp-33 to Asn-41 and the switch II region from residue Gly-63 to Asn-79. For details, refer to the text under “Experimental Procedures” and “Results.”
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Figure 6: Steady state heteronuclear NOE for the backbone amides of rRheb in its GDP- (A) and Gpp(NH)p-bound (B) states. Residues for which no results are shown correspond to either prolines or residues for which relaxation data could not be extracted. The switch I region extends from residue Asp-33 to Asn-41 and the switch II region from residue Gly-63 to Asn-79. For details, refer to the text under “Experimental Procedures” and “Results.”

Mentions: The heteronuclear NOE data in Fig. 6 clearly show that the switch I and, to a lesser extent, the switch II region of GDP-bound Rheb is flexible in solution due to genuine mobility rather than a lack of experimental restraints. In addition, the turn including Pro-113 has values less than 0.6 for the heteronuclear NOE, which is a clear indication of increased flexibility on the pico- to nanosecond time scale and also observed for the C-terminal residues Asp-171 to Ala-174. Obviously, these regions undergo motions faster than the overall tumbling time of the protein. In the 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of Rheb bound to Gpp(NH)p (the nonhydrolyzable analog of GTP), the resonance signals for the switch I (residues Val-32 to Phe-43) and II (residues Asp-60 to Ile-78) regions were broadened beyond detection (Fig. 6B), presumably due to a chemical exchange at an intermediate rate on the NMR time scale, in marked contrast to Rheb-GDP. Interestingly, this difference between the GDP- and Gpp(NH)p-bound forms of Rheb could not directly be inferred from the crystal structure (55). Due to absent resonances in Rheb-Gpp(NH)p, no quantitative differences could be extracted for the switch I and switch II regions.


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)

Steady state heteronuclear NOE for the backbone amides of rRheb in its GDP- (A) and Gpp(NH)p-bound (B) states. Residues for which no results are shown correspond to either prolines or residues for which relaxation data could not be extracted. The switch I region extends from residue Asp-33 to Asn-41 and the switch II region from residue Gly-63 to Asn-79. For details, refer to the text under “Experimental Procedures” and “Results.”
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Steady state heteronuclear NOE for the backbone amides of rRheb in its GDP- (A) and Gpp(NH)p-bound (B) states. Residues for which no results are shown correspond to either prolines or residues for which relaxation data could not be extracted. The switch I region extends from residue Asp-33 to Asn-41 and the switch II region from residue Gly-63 to Asn-79. For details, refer to the text under “Experimental Procedures” and “Results.”
Mentions: The heteronuclear NOE data in Fig. 6 clearly show that the switch I and, to a lesser extent, the switch II region of GDP-bound Rheb is flexible in solution due to genuine mobility rather than a lack of experimental restraints. In addition, the turn including Pro-113 has values less than 0.6 for the heteronuclear NOE, which is a clear indication of increased flexibility on the pico- to nanosecond time scale and also observed for the C-terminal residues Asp-171 to Ala-174. Obviously, these regions undergo motions faster than the overall tumbling time of the protein. In the 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of Rheb bound to Gpp(NH)p (the nonhydrolyzable analog of GTP), the resonance signals for the switch I (residues Val-32 to Phe-43) and II (residues Asp-60 to Ile-78) regions were broadened beyond detection (Fig. 6B), presumably due to a chemical exchange at an intermediate rate on the NMR time scale, in marked contrast to Rheb-GDP. Interestingly, this difference between the GDP- and Gpp(NH)p-bound forms of Rheb could not directly be inferred from the crystal structure (55). Due to absent resonances in Rheb-Gpp(NH)p, no quantitative differences could be extracted for the switch I and switch II regions.

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.

Show MeSH