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Phosphorylated Rho–GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras–GTP to regulate cell migration

Abstract

mTORC2 plays critical roles in metabolism, cell survival and actin cytoskeletal dynamics through the phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae (Dictyostelium discoideum) cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho–GDP promotes AKT phosphorylation by assembling a supercomplex with Ras–GTP and mTORC2. This supercomplex formation is controlled by the chemoattractant-induced phosphorylation of Rho–GDP at S192 by GSK-3. Furthermore, Rho–GDP rescues defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of S192. Thus, in contrast to the prevailing view that the GDP-bound forms of G proteins are inactive, our study reveals that mTORC2–AKT signalling is activated by Rho–GDP.

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Fig. 1: RacE–GDP functions in directed cell migration.
Fig. 2: RacE–GDP specifically interacts with mTORC2.
Fig. 3: RacE–GDP promotes chemoattractant-induced, mTORC2-mediated AKT phosphorylation in cells.
Fig. 4: GSK-3 phosphorylates RacE at S192 in response to chemoattractant.
Fig. 5: S192 phosphorylated RacE–GDP activates mTORC2.
Fig. 6: Phosphorylated RacE–GDP activates mTORC2 in vitro.
Fig. 7: S192 phosphorylated RacE–GDP forms a supercomplex with Tor and Ras–GTP.

Data availability

Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD014014 and provided in Supplementary Tables 1 and 2. Unprocessed images of all blots and gels are provided in Supplementary Fig. 8. The source data for all graphical representations and statistical descriptions are provided in Supplementary Table 5 for Figs. 1b,e, 2a,b,d,h, 3b,d,f,h,j, and 5b,d,f,h and Supplementary Figs. 2 and 6c. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the members of the Iijima and Sesaki labs for their helpful discussions and technical assistance and P. Devreotes and T. Inoue for providing the plasmids and cell lines. This work was supported by grants to M.I. (NIH GM131768 and Allegheny Health Network-Sidney Kimmel Comprehensive Cancer Center), H. Senoo (Japan Society for the Promotion of Science) and H. Sesaki (NIH GM089853).

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Contributions

H. Senoo, H. Sesaki and M.I. conceived the project and designed the study. H. Senoo and M.I. performed the experiments. R.K. assisted with the experiments. S.S., A.N. and Y.K. provided valuable reagents and equipment. H. Senoo, H. Sesaki and M.I. wrote the manuscript.

Corresponding author

Correspondence to Miho Iijima.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Nomenclature.

a, Rho family GTPases in Dictyostelium and human cells. b, Phylogenetic analysis of human RhoA and Rac1 against the Dictyostelium Rho family GTPases. c, Nomenclature of mTORC2 subunits in mammalian, Dictyostelium, and yeast cells.

Supplementary Figure 2 Random cell migration in WT and RacE-KO cells.

Random cell migration was recorded using a Zeiss Axio Vision inverted microscope and analysed by NIH ImageJ (n=15 cells). Values are average ± SD. Statistical analysis was performed using unpaired, two-tailed Student’s t-test.

Supplementary Figure 3 Proteomic identification of RacE binding proteins.

a and b, We identified 94 unique peptides of Tor using mass spectrometry of RacE-binding proteins (46% coverage, 200 unique spectra, 411 total spectra) in a and 10 unique of PiaA peptides (8% coverage, 18 unique spectra, 23 total spectra) in b. The sequences of the identified peptides are highlighted in yellow. Source data for identified peptides for Tor and PiaA are provided in Supplementary Table 1. Source data for all identified proteins are described in Supplementary Table 2.

Supplementary Figure 4 Detection of chemoattractant-induced phosphorylation of PKBA and PKBR1 by immunoblotting with anti-phospho-RPS6KB1 and anti-phospho-PKC-pan antibodies.

a, Amino acid sequences around hydrophobic motif undergoing phosphorylation in PKBA (T435) and PKBR1 (T470) in Dictyostelium. Conserved amino acids in AKT1 and S6K1 (RPS6KB1) in human are highlighted in blue. b, Amino acid sequences around activation loop undergoing phosphorylation in PKBA (T278) and PKBR1 (T309) in Dictyostelium. Conserved amino acids in AKT1 and PKC (PRKCs) in human are highlighted in blue. c, WT, PkbA-KO, and PkbR1-KO cells analysed by immunoblotting with anti-phospho-RPS6KB1 antibodies, anti-phospho-PKC-pan antibodies and antibodies to PKBA and PKBR1 before (0 sec) and after (30 sec) stimulation by the chemoattractant cAMP. Experiments were repeated independently two times with similar results.

Supplementary Figure 5 Comparable expression levels of different GFP–RacE proteins.

WT and RacE-KO cells expressing different RacE-constructs were analysed by immunoblotting with antibodies to GFP and actin. Experiments were repeated independently two times with similar results.

Supplementary Figure 6 Analysis of guanine nucleotide binding to RacE and RasC in response to the chemoattractant cAMP.

a-c, Differentiated Dictyostelium cells carrying GFP–RacE or FLAG–RasC were metabolically labelled using P32 for 1 hour. Cells were lysed after the chemoattractant cAMP for the indicated amounts of time. GFP–RacE and FLAG–RasC were immunopurified using GFP-Trap beads and anti-FLAG beads, respectively. Amount of purified proteins are analysed using SDS–PAGE and CBB staining in a. Bound guanine nucleotides were eluted and analysed by TLC and phosphoimaging in b. Quantification of GTP and GDP signals is shown in c (n=3 independent experiments). Values are the average ± SD.

Supplementary Figure 7 Purification of mTORC2, Tor, RacE, and RasC/G.

a, Immunobloting shows endogenous PiaA was co-purified with FLAG–Tor under CHAPS-containing, low-salt conditions. Untransfected cells were negative controls. b, Anti-FLAG beads carrying FLAG–Tor immunopurified under CHAPS-containing, low-salt conditions were rinsed with 0.5 M NaCl (high-salt) wash after which PiaA dissociated from FLAG–Tor. c, Lst8, detected by mass spectrometry, was also dissociated from FLAG–Tor after the high-salt wash. 18 unique peptides of Lst8 were identified (82% coverage, 44 unique spectra, 86 total spectra), sequences highlighted in yellow. d, PiaA is normally expressed in Rip3-KO cells. WT and Rip3-KO cells were analysed using anti-PiaA antibodies. e and f, The indicated purified proteins were analysed using SDS–PAGE followed by CBB staining. FLAG–Tor, FLAG–RacE proteins, FLAG–RasC/G proteins, FLAG–Lst8, and FLAG–PiaA are indicated (*). Degradation products of FLAG-RasG proteins were observed, detected by anti-FLAG antibodies. Experiments were repeated independently two times with similar results in a, b and d-f.

Supplementary Figure 8

Unprocessed images of all gels and blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table titles/legends

Reporting Summary

Supplementary Table 1

Identified peptides for Tor and PiaA.

Supplementary Table 2

Identified RacE-binding proteins.

Supplementary Table 3

List of plasmids used.

Supplementary Table 4

List of antibodies used.

Supplementary Table 5

Statistics source data.

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Senoo, H., Kamimura, Y., Kimura, R. et al. Phosphorylated Rho–GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras–GTP to regulate cell migration. Nat Cell Biol 21, 867–878 (2019). https://doi.org/10.1038/s41556-019-0348-8

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