Structural basis for the recruitment and selective phosphorylation of Akt by mTORC2.
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| Title: | Structural basis for the recruitment and selective phosphorylation of Akt by mTORC2. |
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| Authors: | Taylor, Martin S., Chen, Maggie, Hancock, Matthew, Wranik, Maximilian, Miller, Bryant D., O'Meara, Timothy R., Palanski, Brad A., Ficarro, Scott B., Groendyke, Brian J., Xiang, Yufei, Kondo, Kazuma T., Linde-Garelli, Karen Y., Lee, Michelle J., Mondal, Dibyendu, Freund, Daniel, Congreve, Samantha, Matas, Kaay, Hennink, Maximiliaan, Xibinaku, Kera, Valenstein, Max L. |
| Source: | Science; 3/5/2026, Vol. 391 Issue 6789, p1-21, 21p |
| Subject Terms: | PROTEIN kinase B, TOR proteins, ENZYME specificity, CELL communication, PROTEIN structure, DRUG target, CELLULAR signal transduction, PROTEIN kinases |
| Abstract: | The mechanistic target of rapamycin (mTOR) protein kinase forms two multiprotein complexes, mTORC1 and mTORC2, that function in distinct signaling pathways. mTORC1 is regulated by nutrients, and mTORC2 is a central node in phosphoinositide-3 kinase (PI3K) and small guanosine triphosphate Ras signaling networks commonly deregulated in cancer and diabetes. Although mTOR phosphorylates many substrates in vitro, in cells, mTORC1 and mTORC2 have high specificity: mTORC2 phosphorylates the protein kinases Akt and PKC, but not closely related kinases that are mTORC1 substrates. To understand how mTORC2 recognizes substrates, we created semisynthetic probes to trap the mTORC2 :: Akt complex and determine its structure. Whereas most protein kinases recognize amino acids adjacent to the phosphorylation site, local sequence contributes little to substrate recognition by mTORC2. Instead, the specificity determinants were secondary and tertiary structural elements of Akt that bound the mTORC2 component mSin1 distal to the mTOR active site and were conserved among at least 18 related substrates. These results reveal how mTORC2 recognizes its canonical substrates and may enable the design of mTORC2-specific inhibitors. Editor's summary: The protein kinase mechanistic target of rapamycin (mTOR) functions in two key signaling complexes, mTORC1 and mTORC2, which phosphorylate distinct substrates. To help clarify the molecular mechanism of such specificity, Taylor et al. used a modified substrate to trap mTORC2 in the process of phosphorylating the protein kinase Akt. Unlike most kinases that recognize features near the residue to be phosphorylated, an adaptor protein in the mTORC2 complex called mSin1 interacted with structural features of Akt some 75 angstroms away from the active site. Structure-based mutagenesis, cross-linking mass spectrometry, modeling, and in vitro functional assays were used to confirm the interaction. Misregulation of mTORC contributes to human disease, so the results may point to new therapeutic strategies. —L. Bryan Ray INTRODUCTION: The protein kinase mechanistic target of rapamycin (mTOR) plays a central role in the control of cell growth, metabolism, and proliferation. Aberrant mTOR signaling contributes to aging, diabetes, and many cancers. By recruiting specific adapter proteins, mTOR forms two mutually exclusive megadalton-scale complexes—mTORC1 and mTORC2—that localize to different cellular membranes and phosphorylate specific substrates as parts of distinct but interconnected signaling networks. Inhibiting mTOR, which suppresses both complexes, is toxic. Selective blockage of individual complexes holds therapeutic promise, yet such strategies remain elusive. mTORC1, which localizes to lysosomes, responds to nutrient and stress signals. Its exclusive Raptor subunit confers substrate specificity by recognizing two distinct linear motifs on canonical substrates such as S6K1 and 4E-BP1. In contrast, mTORC2 is activated at the plasma membrane by extracellular signals such as insulin, other growth factors, and oncogenic Ras to activate target protein kinase substrates including Akt, PKC, and SGK by phosphorylation of their conserved C-terminal hydrophobic motif. RATIONALE: The mechanism underlying mTORC2 substrate recognition remains unknown. Structural studies of the mTORC2 :: Akt interactions have been hindered by the transient nature of kinase-substrate interactions, and in vitro studies were limited by a lack of homogeneous Akt substrates. Here, we used a protein chemical approach called expressed protein ligation to selectively ligate a recombinant protein fragment with a synthetic peptide, making a standard peptide bond. This method enabled us to generate various "semisynthetic" Akt forms as substrates and standards to quantitatively assess mTORC2 activity and as rational probes to trap the mTORC2 :: Akt interaction. RESULTS: Enzymological analysis of mTORC2 phosphorylation of the Akt C-tail confirmed that purified mTORC2 directly and efficiently phosphorylates Akt hydrophobic motif Ser473 and upstream "turn motif" Thr450. Flanking residues were not important for Ser473 phosphorylation, and thus mTORC2 likely recognizes substrates through long-range interactions over local sequences. Cryo–electron microscopy analysis of the mTORC2 :: Akt complex, trapped with the semisynthetic probe Akt-Torin, revealed two critical long-range interactions: (i) The basic surface of the Akt kinase domain N-lobe docks to the acidic loop in the CRIM domain of mTORC2 component mSin1, and (ii) the Akt kinase domain C-lobe interacts with a region within mTORC2 that we term the "mSin1 N-mooring," comprising the N-terminal regions of mTORC2 components mSin1 and Rictor. The same Akt N-lobe residues that bind CRIM also make a stabilizing intramolecular interaction with phospho-Thr450, and thus Akt must undergo a conformational change in interacting with mTORC2. mTORC2 substrates PKC and PKN use the same conserved docking mode. Mutagenesis, together with enzymological and cellular experiments, confirmed these Akt-mTORC2 interaction surfaces, which were also corroborated by cross-linking mass spectrometry analysis. Computational integrative modeling of the experimental data allowed modeling of the mTORC2-Akt interaction at the plasma membrane, revealing how the PH domains of Akt and mTORC2 concurrently engage membrane lipids. CONCLUSION: Our studies uncover the structural basis of mTORC2-catalyzed phosphorylation of Akt and related protein substrates at the plasma membrane and explain the molecular mechanisms that underlie the distinct substrate specificities of mTORC1 and mTORC2. The long-range recruitment surfaces in the mTORC2 :: Akt binding interface may pave the way to selective therapeutic targeting of mTORC2 versus mTORC1. Analogous use of semisynthetic protein-inhibitor conjugates may facilitate the structural determination of other kinase-protein substrate complexes. Structure of mTORC2 recognizing Akt, trapped using the Akt-Torin protein-inhibitor conjugate probe.: mTORC2 recognizes the three-dimensional structure of Akt at two docking interfaces, both ~75 Å distal to the mTOR active site. The Akt kinase N-lobe surface recognized by mTORC2 is the same surface that intramolecularly stabilizes "turn motif" phospho-Thr450. A conformational change enables binding, and then the long, flexible C-tail accesses the mTOR active site. [ABSTRACT FROM AUTHOR] |
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| Database: | Complementary Index |
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