1. Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt
- Author
-
Yu, Zanlin, Chen, Junliang, Takagi, Enzo, Wang, Feng, Saha, Bidisha, Liu, Xi, Joubert, Lydia-Marie, Gleason, Catherine E, Jin, Mingliang, Li, Chengmin, Nowotny, Carlos, Agard, David, Cheng, Yifan, and Pearce, David
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Cardiovascular ,Emerging Infectious Diseases ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Humans ,Immediate-Early Proteins ,Mechanistic Target of Rapamycin Complex 1 ,Monomeric GTP-Binding Proteins ,Phosphorylation ,Protein Serine-Threonine Kinases ,Proto-Oncogene Proteins c-akt ,Rapamycin-Insensitive Companion of mTOR Protein ,Sirolimus ,Transcription Factors ,Akt ,SGK1 ,conformation change ,cryo-EM ,mTORC2 ,structure ,substrate specificity ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.
- Published
- 2022