Dataset Information

Structural insights into Ras regulation by SIN1.

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.

SUBMITTER: Zheng Y

PROVIDER: S-EPMC9171633 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Structural insights into Ras regulation by SIN1.

Zheng Yuyuan Y Ding Lei L Meng Xianhui X Potter Meg M Kearney Alison L AL Zhang Jie J Sun Jie J James David E DE Yang Guang G Zhou Chun C

Proceedings of the National Academy of Sciences of the United States of America 20220506 19

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 abolis ...[more]

PMID: 35522713

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Project description:Inhibitory neurotransmission plays a fundamental role in the central nervous system, with about 30-50% of synaptic connections being inhibitory. The action of both inhibitory neurotransmitter, gamma-aminobutyric-acid (GABA) and glycine, mainly relies on the intracellular Cl- concentration in neurons. This is set by the interplay of the cation chloride cotransporters NKCC1 (Na+, K+, Cl- cotransporter), a main Cl- uptake transporter, and KCC2 (K+, Cl- cotransporter), the principle Cl- extruder in neurons. Accordingly, their dysfunction is associated with severe neurological, psychiatric, and neurodegenerative disorders. This has triggered great interest in understanding their regulation, with a strong focus on phosphorylation. Recent structural data by cryogenic electron microscopy provide the unique possibility to gain insight into the action of these phosphorylations. Interestingly, in KCC2, six out of ten (60%) known regulatory phospho-sites reside within a region of 134 amino acid residues (12% of the total residues) between helices α8 and α9 that lacks fixed or ordered three-dimensional structures. It thus represents a so-called intrinsically disordered region. Two further phospho-sites, Tyr903 and Thr906, are also located in a disordered region between the ß8 strand and the α8 helix. We make the case that especially the disordered region between helices α8 and α9 acts as a platform to integrate different signaling pathways and simultaneously constitute a flexible, highly dynamic linker that can survey a wide variety of distinct conformations. As each conformation can have distinct binding affinities and specificity properties, this enables regulation of [Cl-] i and thus the ionic driving force in a history-dependent way. This region might thus act as a molecular processor underlying the well described phenomenon of ionic plasticity that has been ascribed to inhibitory neurotransmission. Finally, it might explain the stunning long-range effects of mutations on phospho-sites in KCC2.

Dataset Information

Structural insights into Ras regulation by SIN1.

Publications

Structural insights into Ras regulation by SIN1.

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