Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt

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. 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. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase, which belongs to the PI3K-related kinase family and is evolutionarily conserved from yeast to human (1Heitman J. Movva N.R. Hall M.N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.Science. 1991; 253: 905-909Crossref PubMed Scopus (1528) Google Scholar, 2Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1211) Google Scholar). mTOR signaling is central to a large array cellular processes, mediating responses to hormones and growth factors, nutrient availability, and extracellular ionic milieu (3Fu W. Hall M.N. Regulation of mTORC2 signaling.Genes (Basel). 2020; 11: 1045Crossref Scopus (60) Google Scholar). mTOR functions in cellular physiology through two structurally and functionally distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (4Saxton R.A. Sabatini D.M. mTOR signaling in growth, metabolism, and disease.Cell. 2017; 169: 361-371Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 5Luo Y. Xu W. Li G. Cui W. Weighing in on mTOR complex 2 signaling: the expanding role in cell metabolism.Oxid. Med. Cell Longev. 2018; 20187838647Crossref Scopus (36) Google Scholar, 6Gaubitz C. Prouteau M. Kusmider B. Loewith R. TORC2 structure and function.Trends Biochem. Sci. 2016; 41: 532-545Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). These two protein complexes share two conserved components, mTOR and mammalian lethal with SEC13 protein 8 (mLST8, also known as GβL) (7Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. et al.GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar), as well as several complex-specific components. mTORC1 distinctively contains regulatory-associated protein of mTOR (Raptor) and proline-rich Akt substrate of 40 kDa (PRAS40) (8Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. et al.mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2330) Google Scholar, 9Wang L. Harris T.E. Roth R.A. Lawrence Jr., J.C. PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.J. Biol. 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Erdjument-Bromage H. et al.Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2134) Google Scholar, 12Sarbassov D.D. Ali S.M. Sengupta S. Sheen J.H. Hsu P.P. Bagley A.F. et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2166) Google Scholar). The function of mTORC2 has received increasing attention during recent years. The representative downstream effectors of mTORC2 are mainly members of the AGC kinase family, including Akt (also known as protein kinase B), PKC, and serum- and glucocorticoid-induced kinases (SGK1 and SGK3) (13Wu Y.T. Ouyang W. Lazorchak A.S. Liu D. Shen H.M. Su B. mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif.J. Biol. 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D. roles and of mammalian target of rapamycin complexes 1 and 2 during cell in 2020; PubMed Scopus Google Scholar, S. B. J. et of mTOR complexes in from with 2 2017; PubMed Scopus Google Scholar, Y. M. J. Li H. et mTORC2 regulates and 2016; Scopus Google Scholar, J.L. W. G. M. novel target for Scopus Google Scholar). In it first and in to its by rapamycin, functional and structural on mTORC1 has mTORC2. and structural of mTORC1 have its structural in at the structural determinants of substrate specificity and rapamycin have been a within mTOR to the the binding binds rapamycin complex with the binding and mTORC1 and H. B. mTOR kinase and PubMed Scopus Google Scholar). mTORC2 structures have been determined by cryo-EM as but multiple structural that confer context-dependent substrate to of structures that the subunits, mTOR and in mTORC1 and that specificity determinants et used with a cryo-EM structure to interactions between the mTOR and mTORC2 components, mSin1 and Rictor Liu M. Y. Li J. Y. D. et structure of human mTOR complex 2018; PubMed Scopus Google Scholar). suggested that of with the in a that binding of as well as that of the and structural et A. S. D. M. et structure of human 2020; PubMed Google a which into the as well as the and interactions of mSin1 with Rictor and that mSin1 not with mTOR but an important between Rictor and of the structure of the mSin1 N-terminal not is particularly important in the of recruitment of substrates. functional and two distinct substrate interaction in one in the N-terminal of mSin1 found to for and not with Akt M. J. D. protein protein interaction with mTORC2 protein complex and is required for of the Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar). of residues within the core of mTORC2-dependent phosphorylation of but not Akt the in the found to required for binding and of of substrates C. mTORC2 targets AGC kinases through J. 2011; PubMed Scopus Google Scholar). The role of the in an H. S. D. et specificity of TOR complex 2 is determined by a of the 2017; PubMed Google Scholar). the N-terminal has not been insights into the N-terminal and have been In we a representative cryo-EM structure of human mTORC2 apo-complex at of with structures in the presence of Akt and SGK1. the assembly that a for structural including of side in multiple The structure the for mTORC2 to rapamycin, side chain interactions through which Rictor the mTOR determinants of substrate preferences of mTORC1 and mTORC2 are also new into the structural for mTORC2 context-dependent of and Akt is in the presence of SGK1, but not Akt, the conformation of the mSin1 N-terminal is in a large rotation of to form a with Rictor The functional of structural is in phosphorylation In to obtain mTORC2 core complex for and of human in The complex by and to binding a new of the Liu Y. Li S. S. Y. et and for high-resolution Sci. S. A. 2020; PubMed Scopus Google Scholar), cryo-EM cryo-EM to and in the apo-state substrates. The of mTORC2 tested and and and used for the and on the a for the mTORC2 apo-state structures for of mTORC2 with two of its Akt and SGK1. on two substrates in of their in and and metabolism, and on and (3Fu W. Hall M.N. Regulation of mTORC2 signaling.Genes (Basel). 2020; 11: 1045Crossref Scopus (60) Google Scholar, M. J. D. protein protein interaction with mTORC2 protein complex and is required for of the Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar, D. and of the 11: PubMed Scopus Google Scholar). The mTORC2 structure of two a to a Liu M. Y. Li J. Y. D. et structure of human mTOR complex 2018; PubMed Scopus Google Scholar). The of the apo-complex is that of a with a and a of and of the of mTOR, and mSin1 with a of The two mTOR form a that as the of the and Rictor in on its two The structures of and mTOR as well as their interaction are to that in mTORC1 H. B. mTOR kinase and PubMed Scopus Google Scholar, E. S. D. Hall M.N. et of human mTOR complex 2016; PubMed Scopus Google Scholar). The kinase of mTOR is in the between and Rictor. as mSin1 a structure and which has an extended that the the mTOR that mTORC2 is not a complex, and the two found to in two mainly the core of the complex in the of the interaction between the two mTOR subunits, as in and In the the two mTOR are to the core of the complex and a conformation to that in mTORC1 The structure of Rictor includes protein and the yeast kinase helical repeat and of which direct contacts with and also with with and cryo-EM Rictor and not but are by In the of mTOR is in to attention to its interaction with Rictor. In is to the kinase and to the N-terminal which a central role in substrate recruitment and H. B. mTOR kinase and PubMed Scopus Google and is in binding the complex E. S. D. Hall M.N. et of human mTOR complex 2016; PubMed Scopus Google Scholar). In the is to the mTOR kinase but also interacts with and of Rictor. of Rictor sterically the from with the complex while in the also mTORC2 interaction with substrates and a interaction H. Li B. M. A. et of mTORC1 by and by 2017; PubMed Scopus Google Scholar). and observed in to to the interaction of Rictor and the through and to the differential of mTORC1 and mTORC2 which to an important functional role in substrate by mTORC1 H. B. mTOR kinase and PubMed Scopus Google Scholar, Lawrence Jr., J.C. The substrate by the mammalian target of Cell Biol. 2002; 22: PubMed Scopus Google Scholar), is by is direct interaction between Finally, Akt on the of Rictor and on functional and mSin1 contains an N-terminal and W. et is for and its distinct Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). For the we the structure of the N-terminal while the are in the to their as has been in Liu M. Y. Li J. Y. D. et structure of human mTOR complex 2018; PubMed Scopus Google Scholar, A. S. D. M. et structure of human 2020; PubMed Google Scholar). one of the the has been in binding of mTORC2 Akt, and The N-terminal contains distinct a a Rictor and and an The includes a pair of short α-helices nestled between and of by an extended strand, which the of multiple weak contacts and which we have the has not been visualized in which is in of the role it in substrate in to for not Akt with and phosphorylation by mTORC2 M. J. D. protein protein interaction with mTORC2 protein complex and is required for of the Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar). In the the side chain is well and to with a interaction the of the we in a negatively charged of the Rictor and specifically with within the which to the substrate for AGC kinase and has been to by Akt and P. W. H. Lazorchak A.S. D. et phosphorylation mTORC2 complex and inhibits downstream Akt to Cell Biol. PubMed Scopus Google however, in the structures it is of the an to and the the mTOR mSin1 in extensive interactions with with a short by an extended which of the the of and the of mSin1 in a short into the In the with Akt and SGK1, the substrates not to and of the as a of with substrates in the after at 8 in to the from not from in the apo-complex it is also that the are of downstream of mSin1, as the not in the In the interaction of the substrates with mTORC2 the and of the to the apo-complex and the of the mSin1 N-terminal with Rictor markedly altered in the the of Arg-83 including to Akt, in the in its presence to the apo-complex the Arg-83 side chain a large rotation a negatively charged patch of Rictor into to with which it to form a and In to the functional role of Arg-83 in differential of and Akt, we tested the of to support mTORC2 kinase activity by Li B. G. et at distinct specificity in of and Cell Sci. PubMed Scopus Google Scholar). 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D. protein protein interaction with mTORC2 protein complex and is required for of the Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar). Here, we that in the the side chain with a we multiple side between and weak interactions with Rictor the side chain of which interacts with the of the and an important for the which the is in that phosphorylation of has been suggested to mTORC2 P. W. H. Lazorchak A.S. D. et phosphorylation mTORC2 complex and inhibits downstream Akt to Cell Biol. PubMed Scopus Google Scholar). however, as have found that phosphorylation of mTORC2 activity G. positive between Akt and mTORC2 via Full Text Full Text PDF PubMed Scopus Google Scholar). The structural of context-dependent of phosphorylation are at time, as in structures and of A. S. D. M. et structure of human 2020; PubMed Google Scholar). of apo-complex and co-complex structures a of on mTORC2 conformation in the Arg-83, which is from residue in the a large rotation a patch in which includes with which it to form a with the of Arg-83 to as it interacts with and from its to Rictor that the new interaction interactions to the of mTORC2 and that the substrate-induced in it to with the in binding and the hydrophobic to the mTOR The functional of Arg-83 is by the that an Ala of that residue selectively disrupts but not Akt In the with Akt and SGK1, the substrates not and an important of is that a in which an co-complex to cryo-EM to that substrate A. S. D. M. et structure of human 2020; PubMed Google Scholar), with of the mSin1 is of to the of structural for mTORC2 of its distinct substrates. Akt, and are mTORC2 however, are differences in the of and (3Fu W. Hall M.N. Regulation of mTORC2 signaling.Genes (Basel). 2020; 11: 1045Crossref Scopus (60) Google Scholar, Li B. G. et at distinct specificity in of and Cell Sci. PubMed Scopus Google Scholar). recent have and effectors that selectively mTORC2 phosphorylation of but not Akt B. P. et through mTOR to its Scholar, Li B. G. et at distinct specificity in of and Cell Sci. PubMed Scopus Google Scholar). Akt is by its N-terminal which binds to with mTORC2 at the and with interaction kinase and substrate C. mTORC2 targets AGC kinases through J. 2011; PubMed Scopus Google Scholar, D. The and of AGC protein 11: Scopus Google Scholar). has a distinct N-terminal which binds but not Li H. J. C. et of and kinase 1 binds to and is essential for J. 2007; PubMed Scopus Google Scholar). with the mSin1 and is essential for its C. mTORC2 targets AGC kinases through J. 2011; PubMed Scopus Google Scholar, M. J. D. protein protein interaction with mTORC2 protein complex and is required for of the Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar). that binding and the conformation in mSin1 an important role in of by Li B. G. et at distinct specificity in of and Cell Sci. PubMed Scopus Google and responses to as E. G. S.H. Y. et of mTORC2 in of in to and Biol. Chem. Full Text Full Text PDF PubMed Google changes in B. P. et through mTOR to its Scholar). to the interactions of with mSin1 that support its by mTORC2. 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