Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation Linda Truebesteina,b, Harald Horneggera,b, Dorothea Anratherc, Markus Hartlc, Kaelin D. Flemingd, Jordan T. B. Starihad, Els Pardone,f, Jan Steyaerte,f, John E. Burked,g, and Thomas A. Leonarda,b,1 aDepartment of Structural and Computational Biology, Max Perutz Labs, Vienna BioCenter, 1030 Vienna, Austria; bDepartment of Medical Biochemistry, Medical University of Vienna, 1090 Vienna, Austria; cMass Spectrometry Core Facility, Max Perutz Labs, Vienna BioCenter, 1030 Vienna, Austria; dDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada; eStructural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium; fVIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium; and gDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada Edited by Natalie G. Ahn, University of Colorado, Boulder, CO, and approved July 11, 2021 (received for review February 1, 2021) The protein kinase Akt is one of the primary effectors of growth reveal an interface of ∼1,500 Å2 of buried surface area (27–29). factor signaling in the cell. Akt responds specifically to the lipid Stoichiometric phosphorylation of the activation loop in combi- second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5) nation with a phosphomimetic aspartate substitution in the hy- P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH do- drophobic motif is insufficient to overcome the requirement for main, leading to phosphorylation of its activation loop and the hy- PI(3,4,5)P3 or PI(3,4)P2 for full activity (20). Accordingly, im- drophobic motif of its kinase domain, which are critical for activity. aging of the Akt-substrate complex in live cells revealed that Akt We have now determined the crystal structure of Akt1, revealing an is active only in its membrane-bound state (23). Within the cell autoinhibitory interface between the PH and kinase domains that is interior, PI(3,4)P has been shown to control Akt signaling on often mutated in cancer and overgrowth disorders. This interface per- 2 sists even after stoichiometric phosphorylation, thereby restricting endosomal membranes (23, 30, 31). Whether PIP3 or PI(3,4)P2 is necessary for sustained Akt ac- maximum Akt activity to PI(3,4,5)P3-orPI(3,4)P2-containing mem- branes. Our work helps to resolve the roles of lipids and phosphory- tivity in the cell, however, is still controversial. It was recently lation in the activation of Akt and has wide implications for the reported that Akt could not be activated by PI(3,4,5)P3 in vitro but that it could be activated independently of lipids by phos- spatiotemporal control of Akt and potentially lipid-activated kinase BIOCHEMISTRY signaling in general. phorylation of its hydrophobic motif (21). The authors proposed that phosphorylation of S473 activates Akt through the formation kinase | signaling | lipid | PIP3 | Akt of an electrostatic interaction with a conserved basic residue (R144) in the PH-kinase domain linker, thereby relieving PH domain- kt is a serine/threonine protein kinase essential for cell mediated autoinhibition. More recently, the mechanism by which Agrowth, proliferation, and metabolism (1, 2). Growth factor S473 phosphorylation induces PH domain displacement and, and insulin signaling via receptor tyrosine kinases activate class I thereby, Akt activation was reported to rely on a conformational phosphoinositide 3-kinase (PI3K), which generates the lipid sec- change in the PH domain (32). In this model, Akt can be activated ond messenger phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5) by phosphorylation, independent of its binding to PI(3,4,5)P3 or P3]. Akt is activated by PIP3 in the membrane and phosphorylates up to 100 substrates in diverse physiological processes (3). Significance Hyperactivation of the PI3K-Akt pathway is frequently observed in human cancers and overgrowth disorders (4), while autosomal Akt is an essential protein kinase that controls cell growth, dominant inactivation of Akt2 leads to insulin resistance and survival, and metabolism. Akt is activated by the lipid second diabetes (5). messengers PIP and PI(3,4)P and by phosphorylation. How- Akt comprises an N-terminal lipid-binding PH domain with 3 2 ever, the relative contributions of lipid binding and phos- specificity for PI(3,4,5)P and phosphatidylinositol-3,4-bisphos- 3 phorylation to Akt activity in the cell are controversial. Here, phate [PI(3,4)P ](6–8) and a C-terminal AGC kinase domain 2 we have determined the structure of autoinhibited Akt1, which characterized by the presence of a regulatory tail at its C terminus reveals how the lipid-binding PH domain maintains the kinase (9). The C-tail of human Akt1 contains two phosphorylation sites, domain in an inactive conformation in the absence of PIP . one in the turn motif (T450) and one in the hydrophobic motif 3 Despite stoichiometric phosphorylation, Akt adopts an auto- (S473). Phosphorylation of the turn motif occurs cotranslationally inhibited conformation with low basal activity in the absence (10), protects Akt from ubiquitin-mediated degradation (10, 11), of PIP . Our work reveals the mechanistic basis of Akt hyper- and is unaffected by growth factor signaling (11). Recruitment of 3 activation in cancer and overgrowth diseases and unambigu- Akt to PI(3,4,5)P in the plasma membrane promotes its phos- 3 ously establishes that Akt depends on lipids for activity in phorylation by phosphoinositide-dependent kinase 1 (PDK1) in its the cell. activation loop (T308) (12, 13) and by mTORC2 in its hydro- phobic motif (14), rendering it active against substrates. S473 Author contributions: L.T., H.H., J.E.B., and T.A.L. designed research; L.T., H.H., K.D.F., phosphorylation promotes a disorder-to-order transition of the J.T.B.S., and T.A.L. performed research; H.H., E.P., and J.S. contributed new reagents/an- hydrophobic motif (15) that, in synergy with activation loop alytic tools; L.T., H.H., D.A., M.H., K.D.F., J.T.B.S., J.E.B., and T.A.L. analyzed data; and phosphorylation and ATP, stabilizes a phosphatase-resistant active T.A.L. wrote the paper. conformation (16–20) that exhibits catalytic activity orders of The authors declare no competing interest. magnitude higher than unphosphorylated Akt (21, 22). Akt lack- This article is a PNAS Direct Submission. ing phosphorylation of these sites is catalytically inactive (23). This open access article is distributed under Creative Commons Attribution License 4.0 Akt exists in an autoinhibited conformation in the cytosol of (CC BY). unstimulated cells, characterized by an intramolecular interac- 1To whom correspondence may be addressed. Email: [email protected]. tion between its PH domain and the substrate-binding cleft of its This article contains supporting information online at https://www.pnas.org/lookup/suppl/ kinase domain (20, 23–26). Crystal structures of C-terminally doi:10.1073/pnas.2101496118/-/DCSupplemental. truncated Akt1 in complex with various allosteric inhibitors Published August 12, 2021. PNAS 2021 Vol. 118 No. 33 e2101496118 https://doi.org/10.1073/pnas.2101496118 | 1of11 Downloaded by guest on September 30, 2021 PI(3,4)P2. The respective roles of PIP3 and phosphorylation in the and mediating essential crystal lattice contacts. Previously repor- control of Akt activity are therefore an open question. ted mutations in Akt1, Akt2, and Akt3 associated with Proteus In order to resolve the molecular mechanisms of Akt activation, Syndrome, several cancers, and megalencephalies (33–38) and we have determined the high-resolution structure of Akt1 in com- mutations in Akt1 that drive growth factor-independent cell sur- plex with a nanobody, detailing the autoinhibitory interface between vival in vitro (39) all map to the autoinhibitory interface (Fig. 1D). the PH and kinase domains. This interface both sequesters the In our structure, the PH domain is rotated ∼23° with respect to the PIP3-binding site of the PH domain and is mutually exclusive with structure of Akt in complex with allosteric inhibitors (27–29), the active conformation of the kinase domain. Rare mutations of leading to an overall root mean square deviation of 9.04 Å over Akt associated with cancer (33) and overgrowth disorders (34–36) the whole PH domain (Fig. 1E and SI Appendix, Fig. S2F), indi- map to this autoinhibitory interface. By combining a spectrum of cating that allosteric inhibitors severely distort the autoinhibitory biophysical, structural, and biochemical techniques, we demonstrate interaction between the PH and kinase domains. In the absence of that, while stoichiometric activation loop and hydrophobic motif the C-terminal 35 amino acids, which comprise the turn motif phosphorylation increase the basal kinase activity of Akt1 in vitro, (T450) and the hydrophobic motif (S473), density was not ob- they do not completely relieve autoinhibition by its PH domain. Our served for the glycine-rich loop (residues 154 to 157), αChelix findings provide clear evidence that full activation of Akt is de- (residues 180 to 196), or the activation loop (residues 289 to 306), pendent on both phosphorylation and lipid binding and, therefore, though inspection of the structure reveals that there are no bar- that its activity against cellular substrates is most likely restricted to riers to the αC helix or the glycine-rich loop from