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Structural insights into Ras regulation by SIN1
Yuyuan Zheng1,2,3, Lei Ding1,2, Xianhui Meng3
1School of Public Health, Zhejiang University School of Medicine, Hangzhou, 310058, China.
Abstract:
Over the years it has been established that SIN1, a key component of mTORC2, could interact with Ras family small GTPases through its Ras-binding domain (RBD). The physical association of Ras and SIN1/mTORC2 could potentially affect both mTORC2 and Ras-ERK pathways. To decipher the precise molecular mechanism of this interaction, we determined the high-resolution structures of HRas/KRas-SIN1 RBD complexes, showing the detailed interaction interface. Mutation of critical interface residues abolished Ras-SIN1 interaction and in SIN1 knockout cells we demonstrated that Ras-SIN1 association promotes SGK1 activity but inhibits insulin-induced ERK activation. With structural comparison and competition fluorescence resonance energy transfer (FRET) assays we showed that HRas-SIN1 RBD association is much weaker than HRas-Raf1 RBD but is slightly stronger than HRas-PI3K RBD interaction, providing a possible explanation for the different outcome of insulin or EGF stimulation. We also found that SIN1 isoform lacking the PH domain binds stronger to Ras than other longer isoforms and the PH domain appears to have an inhibitory effect on Ras-SIN1 binding. In addition, we uncovered a Ras dimerization interface that could be critical for Ras oligomerization. Our results advance our understanding of Ras-SIN1 association and crosstalk between growth factor-stimulated pathways.
Insights
The study reveals how SIN1 (stress-activated protein kinase-interacting protein 1) binds to Ras proteins, impacting cell signaling pathways. This interaction influences cell growth and survival by modulating mTORC2 and Ras-ERK pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Structural Biology
Background:
- SIN1 is a key component of mTORC2, known to interact with Ras GTPases via its Ras-binding domain (RBD).
- The physical association between Ras and SIN1/mTORC2 may influence both mTORC2 activity and the Ras-ERK pathway.
- Understanding this interaction is crucial for deciphering crosstalk between growth factor-stimulated pathways.
Purpose of the Study:
- To determine the high-resolution structures of HRas/KRas-SIN1 RBD complexes.
- To elucidate the precise molecular mechanism of Ras-SIN1 interaction.
- To investigate the functional consequences of Ras-SIN1 association on cellular signaling.
Main Methods:
- High-resolution structural determination of HRas/KRas-SIN1 RBD complexes.
- Site-directed mutagenesis of critical interface residues.
- Experiments in SIN1 knockout cells.
- Competition fluorescence resonance energy transfer (FRET) assays.
- Structural comparisons with other Ras-binding proteins.
Main Results:
- Detailed interaction interfaces between Ras and SIN1 RBD were revealed by structural analysis.
- Mutations at the interface disrupted Ras-SIN1 binding.
- Ras-SIN1 association was shown to promote SGK1 activity and inhibit insulin-induced ERK activation in SIN1 knockout cells.
- HRas-SIN1 RBD binding affinity was quantified relative to HRas-Raf1 RBD and HRas-PI3K RBD.
- SIN1 isoforms lacking the PH domain exhibited stronger Ras binding, suggesting an inhibitory role for the PH domain.
Conclusions:
- The study provides atomic-level insights into Ras-SIN1 interaction and its functional implications.
- Ras-SIN1 association plays a role in modulating SGK1 activity and ERK pathway signaling.
- The binding affinity and regulation by SIN1 isoforms offer explanations for differential pathway activation.
- A novel Ras dimerization interface was identified, potentially involved in Ras oligomerization.
- These findings advance the understanding of signal integration and crosstalk in growth factor signaling pathways.