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LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control

Abstract

The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.

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Fig. 1: The Hippo pathway negatively regulates mTORC1 signalling via the LATS1/2 kinases.
Fig. 2: The LATS1/2 kinases bind and phosphorylate Raptor.
Fig. 3: The Raptor-S606D mutation inhibits growth factor-induced mTORC1 activation.
Fig. 4: Raptor-S606 phosphorylation inversely regulates the interaction of Raptor with Rheb and DEPTOR.
Fig. 5: The Raptor-S606D mutation suppresses glycolysis and lipid biosynthesis.
Fig. 6: The Raptor-S606D mutation suppresses cell growth and proliferation.
Fig. 7: Phosphorylation of Raptor-S606 reduces mTORC1 activity and organ size in mouse.

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Data availability

All data that support the findings of this study are available on request from the corresponding author.

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Acknowledgements

We thank B. North, B. Wang, F. Dang, C. Jiang, Y. Gao, N.T. Nihira, K. Shimizu, H. Inuzuka, J. Zhang and other Wei laboratory members for their critical reading and discussion of the manuscript. We also thank B. Manning (Harvard T. H. Chan School of Public Health) for helpful suggestions and providing HEK293E cells, the 4E-BP1 construct and LAMP2 antibody. The LATS1/2-dKO, MST1/2-dKO, 3KO and 8KO HEK293A cells were a gift from K.-L. Guan (UC San Diego). X.D. and J.G. were supported by NRSA T-32 training grants. P.L. is in part supported by grant no. R00CA181342 from the National Cancer Institute. Y.L. and Y.Y. are supported by NCI grant no. R01CA222571. The AAV virus work was partially supported by NIH grant no. P30EY012196. The LC–MS/MS work was partially supported by NIH grants nos P01CA120964 (J.A.) and P30CA006516 (J.A.). The remainder work was supported in part by pilot funding from the MUSC Digestive Diseases Research Core Center Pilot and Feasibility Program (W.G.) and NIH grants (grant nos R00CA207867 (W.G.), R01GM094777 (W.W.) and R01CA177910 (W.W.)).

Author information

Authors and Affiliations

Authors

Contributions

W.G. and W.W. designed the experiments. W.G. and Xiaoming Dai performed the experiments with assistance from Xiangpeng Dai, S.Y., J.G., M.W., J.L. J.H., R.J.Q., N.J.G. and P.L. J.M.A. performed the LC–MS/MS metabolomic profiling and mass-spectrometry analysis of Raptor-S606 phosphorylation. J.X., J.Z., C.W., Y.L., Y.Y, Z.H. and G.G. helped to design and perform the AAV-mediated depletion of Nf2 and Lats1/2 experiments. W.W. and P.P.P. supervised the study. W.G. and W.W. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Wenjian Gan or Wenyi Wei.

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Extended data

Extended Data Fig. 1 Activation of the Hippo pathway negatively regulates mTORC1 kinase activity independent of YAP/PTEN.

a–d, HEK293, HEK293E (a), HEK293 A (b), MCF7 (c) and HeLa (d) cells were cultured in 60 mm dishes at 25%, 50%, 75% and 100% confluence for 16 h before harvesting for immunoblot (IB) analysis. e, Increasing cell density activates LATS1 in vitro. IB analysis of LATS1 in vitro kinase assay samples. f, HEK293 cells were seeded on 60 mm dishes at 30% (L), 60% (M) and 100% (H) confluence for 16 h and then replaced with fresh medium for 1 hour. The media was collected for Mass spectrometry analysis of relative amino acid (AA) levels. Data were shown as mean ± s.d. of n = 3 biological independent samples. g, IB analysis of whole cell lysates (WCLs) derived from HEK293 cells treated with 1 μg/ml LatB or 10 μM FSK for indicated time. h, HEK293 wild type (WT) and PTEN knockout (PTEN-KO) cells were cultured in 60 mm dishes at 30% (L) and 100% (H) confluence for 16 hours before harvesting for IB analysis. i,j, IB analysis of WCLs derived from WT and PTEN-KO HEK293 cells treated with 1 μg/ml LatB or 10 μM FSK for 60 min. k–m, HEK293 cells were infected with lentiviral shYAP and shTAZ vectors and then selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells. The resulting cells were cultured in 60 mm dishes at 30% (L) and 100% (H) confluence for 16 hours (k) or treated with 1 μg/ml LatB (l) or 10 μM FSK (m) for 60 min before harvesting for IB analysis. Western blots in ae and gm were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 1. Statistical source data are available in Statistical Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 The Hippo pathway suppresses mTORC1 signalling depending on LATS1/2 kinase activity.

a,b, IB analysis of WCLs derived from HEK293A-WT, MAP4K4/6/7 (3KO) and MAP4K1/2/3/4/6/7 MST1/2 (8 KO) cells treated with 10 μM FSK (a) or 1 μg/ml LatB (b) for 60 min. c,d, IB analysis of WCLs derived from HEK293-WT and SAV1-KO cells at 30% (L) and 100% (H) confluence (c) or treated with 1 μg/ml LatB (d) for 60 min. e, IB analysis of WCLs derived from 293-WT and NF2-KO cells treated with 1 μg/ml LatB for 60 min. f, IB analysis of WCLs derived from 293 cells transfected with LATS1/MST1 or EV (empty vector, as a negative control). The cells were treated with 1 μM LPA or 1 μM S1P for 60 min before harvesting. g, LATS1/2 dKO cells were infected with EV, WT-LATS1 or kinase-dead LATS1 (AA) vector and selected with 200 μg/ml hygromycin for 72 hours to eliminate the non-infected cells. The resulting cells were cultured in 60 mm dishes at 30%, 60% and 100% confluence and harvested for IB analysis. h, In vitro kinase assays demonstrate that genetic deletion of LATS1/2 leads to increased mTORC1 kinase activity in vitro towards phosphorylating bacterially purified 4E-BP1. Raptor immunoprecipitates (IP) were prepared from HEK293A-WT or LATS1/2 dKO cells and used for examination of mTORC1 kinase activity in vitro. 250 nM mTOR inhibitor Torin 1 was added as indicated. Western blots in ah were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 LATS1/2 interacts with and phosphorylates Raptor at S606.

a, A schematic illustration of the Raptor domain structures. b, The MH domain of Raptor interacts with endogenous LATS1. IB analysis of WCLs and GST pull-down (GST-PD) products derived from HEK293 cells transfected with indicated constructs. c, The MS/MS fragmentation spectrum showing distinct b- (N-terminal) and y- (C-terminal) series fragment ions for the Raptor peptide LYpSLLSDPIPEVR defining the pS606 site. This experiment was performed one time. d, Protein sequence illustration of synthesized Raptor peptides. e, Titration of the indicated Raptor peptides with or without S606 phosphorylation demonstrated that the generated Raptor-pS606 antibody specifically recognizes the pS606 epitope. f, S606A mutation blocks LATS1-mediated Raptor-S606 phosphorylation. IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. g,h, LATS1/2 dKO blocks Raptor-S606 phosphorylation. IB analysis of WCLs derived from HEK293-WT, LATS1/2 dKO cells treated with 1 μg/ml LatB (g) or 10 μM FSK (h) for 60 min. i,j, LATS is the major kinase phosphorylating Raptor at S606. HEK293 cells were infected with lentiviral vectors targeting indicated kinases and then selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells. LATS1/2 KO cells as a positive control. k, S606D mutation decreases mTORC1 kinase activity in vitro. HA-Raptor IP were prepared from HEK293 cells and used for examination of mTORC1 kinase activity. 250 nM mTOR inhibitor Torin 1 was added as indicated. Western blots in b and e-k were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Generation of Raptor S606Dknock-in and S606Aknock-in cells by CRISPR.

a, A schematic representation of the amino sequence and strategy to generate Raptor-S606D and S606A CRISPR knock-in cells. b, Identification of the potential knock-in mutants. Genomic DNA containing Raptor-S606D or S606A mutation were amplified by PCR and digested with SfcI. The experiment was repeated three times independently, with similar results obtained. c, Confirmation of the correct mutation of Raptor-S606D or S606A by Sanger DNA sequencing. d,e, S606Dknock-in cells display reduced mTORC1 activity. IB analysis of WCLs derived from HEK293 (d) and HEK293E (e) S606Dknock-in and S606Aknock-in cells. f,g, The S606A mutation largely blocks LatB or FSK-induced Raptor-pS606. IB analysis of WCLs derived from HEK293 knock-in cells treated with 1 μg/ml LatB (f) or 10 μM FSK (g) for indicted time periods. h, In vitro kinase assays shows that the S606D mutation reduces mTORC1 kinase activity in vitro towards phosphorylating S6K1. Raptor immunoprecipitates (IP) derived from HEK293-WT or S606Dknock-in cells were analysed for mTORC1 kinase activity. 250 nM mTOR inhibitor Torin1 was added as indicated. i, IB analysis of WCL derived from HEK293-WT and S606Dknock-in cells transfected with HA-YAP1. j, IB analysis of WCLs derived from HEK293-WT and S606Aknock-in cells infected with shYAP/TAZ virus. Western blots in d-j were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 LATS1 suppresses Rheb- and growth factor-induced mTORC1 activation.

a,b, Growth factor-induced activation of mTORC1 is impaired in S606Dknock-in cells. IB analysis of WCLs derived from HEK293 or HEK293E cells treated with 100 nM insulin (a) or 100 ng/ml IGF-1 (b). c, IB analysis of WCLs derived from Rheb-depleted HEK293 cells treated with 100 nM insulin. Cells were serum-starved for 16 hours before insulin stimulation. d, Rheb-WT and the GTP-bound mutant Q64L activate mTORC1 in vivo. IB analysis of WCLs derived from HEK293 cells transfected with indicated Rheb constructs. e, LATS1-WT inhibits Rheb-induced activation of mTORC1. IB analysis of WCLs derived from HEK293 cells transfected with indicated constructs. f, LATS1 does not affect RagB-induced activation of mTORC1. IB analysis of WCLs derived from Rap2A or RagB-Q99L expressing HEK293 cells transfected with indicated constructs. g, Depletion of DEPTOR cannot restore mTORC1 activity in S606Dknock-in cells. IB analysis of WCLs derived from HEK293E-WT and S606Dknock-in cells infected with lentiviral shDEPTOR vectors. h, The S606D mutation inhibits mTORC1 activation in TSC2-depleted cells under serum-starved condition. IB analysis of WCLs derived from HEK293E-WT, S606Dknock-in and S606Aknock-in cells infected with lentiviral shTSC2 vector. Cells were serum-starved for 16 hours before harvesting. i, LatB suppresses Tsc2-loss-induced mTORC1 activation. IB analysis of WCLs derived from Tsc2+/+ and Tsc2-/- MEFs treated with 1 μg/ml LatB for indicated time period under serum-starved condition. j, Rheb activates mTORC1 in vitro in a GTP loading-dependent manner. HA-Raptor immunoprecipitates derived from HEK293 cells were analysed for mTORC1 kinase activity toward 4E-BP1 in the presence of GTP- or GDP-bound Rheb. k, A proposed model to describe mechanistically how LATS1/2-mediated Raptor-S606 phosphorylation inhibits growth factors-induced mTORC1 activation. Western blots in aj were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 The Raptor-S606D mutation affects its interaction with Rheb and DEPTOR in a LATS-dependent manner.

a,b, The Raptor-S606D mutation does not affect mTORC1 complex formation. IB analysis of WCLs and IP derived from HEK293 CRISPR knock-in cells (a) or HEK293 cells transfected with indicated constructs (b). c, The S606D mutation impairs its interaction with Rheb. IB analysis of WCLs and IP derived from indicated CRISPR knock-in cells expressing HA-tag Rheb. d, The Raptor-S606D mutation does not affect its interaction with Rag GTPases. IB analysis of WCLs and IP derived from indicated HEK293 CRISPR knock-in cells. e, The Raptor-S606D mutation increases its interaction with DEPTOR. IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. f, The Raptor-S606D mutation minimally affects its interaction with PRAS40. IB analysis of WCLs and IP derived from indicated HEK293 CRISPR knock-in cells. g, IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. h, IB analysis of WCLs and GST pull-down products (GST-PD) derived from HEK293 cells transfected with indicated constructs. i,j, IB analysis of WCLs and IP derived from indicated HEK293 CRISPR knock-in cells stably expressing HA-Rap2A (as a negative control) or HA-Rheb. The cells were treated with 1 μg/ml LatB (i) or 10 μM FSK (j) for 60 min before harvesting. k, IB analysis of WCLs and IP derived from HEK293 cells stably expressing HA-Rheb or HA-DEPTOR cultured at low (30%) or high (100%) cell density. l,m, IB analysis of WCL and IP derived from indicated HEK293 CRISPR knock-in cells. The cells were treated with 1 μg/ml LatB (l) or 10 μM FSK (m) for 60 min before harvesting. Western blots in a–m were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 Rag GTPases, PRAS40, Rheb and DEPTOR bind different domains of Raptor.

ac, Rag GTPases and PRAS40 interact with Raptor N-terminus. IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. d, DEPTOR binds the MH domain and C-terminus of Raptor. IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. e, A schematic representation of the indicated domains of Raptor and a summary of each domain that is responsible for binding different Raptor-interacting proteins. f, Rheb does not affect the interaction between Raptor and PRAS40. IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. g,h, IB analysis of WCLs and IP derived from HEK293 cells transfected with indicated constructs. Where indicated, cells were treated with 10 nM mTOR inhibitor Torin 1 for 1 hour before harvesting. Western blots in ad and fh were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 7.

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Extended Data Fig. 8 The Raptor S606D mutation inhibits cell proliferation, colony formation and tumorigenesis.

a, S606Aknock-in cells display bigger cell size than WT cells at high cell confluence. Cells were grown in 10% serum at 25% or 90% confluence and subjected to cell size analysis by FACS. b, Growth curve of HEK293-WT, S606Dknock-in and S606Aknock-in cells to show that S606Dknock-in cells proliferate slowly compared with WT and S606Aknock-in cells. c, S606Dknock-in cells, but not S606Aknock-in cells, display reduced colony formation ability (bottom). Data were shown as mean ± s.d. of three independent experiments. P values were calculated using two-tailed Student’s t-test. d, IB analysis of WCLs derived from parental and Raptor knockout (Raptor-KO) cells. Blot is representative of two independent experiments, with similar results obtained. e,f, Colony formation assays (e) and soft-agar assays (f) demonstrate the deficiency of Raptor-KO cells on cell transforming ability. Data were shown as mean ± s.d. of three independent experiments. P values were calculated using two-tailed Student’s t-test. g, Tumours derived from xenograft assay in were dissected (n = 4 mice). Unprocessed immunoblots are shown in Source Data Extended Data Fig. 8. Statistical source data are available in Statistical Source Data Extended Data Fig. 8.

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Extended Data Fig. 9 Generation and characterization of RaptorD/D knock-in mice.

a, sgRNA sequence and part of ssODN sequence used for generating RaptorD/D mice. b, Analysis of mouse genomic DNA by SfcI digestion. c, Sanger sequencing results of RaptorD/D mouse genomic DNA. d. Frequency of genotypes produced from Raptor+/D mouse intercrosses. e, Representative images of 2-week-old and 8 week-old mice. f, Body weight of mice. Data were shown as mean ± s.e.m. (n = 44 mice/group). g, Growth of female and male mice. Data were shown as mean ± s.e.m. (female, n = 20 mice/group; male, n = 18 mice/group). h, Body weight of mice fed with high fat diet (HFD). Data were shown as mean ± s.e.m. (n = 7 mice/group). i, Representative images of Raptor+/+ and RaptorD/D mice fed with HFD for 11 weeks. j, Weight of white adipose tissue (WAT), brown adipose tissue (BAT) and liver was shown as percentage of body weight in mice that were on an HFD for 11 weeks. Data were shown as mean ± s.e.m. (n = 7/group). k, Representative image of bodipy staining of liver sections from Raptor+/+ and RaptorD/D mice that were on an HFD for 11 weeks. l, Organ sections were analysed by immunohistochemistry for pS6 (pS235/S236) levels. Scale bars, 50 μm for spleen and 100 μm for brain. m, IB analysis of WCLs derived from indicated organs. n, IB analysis of WCLs derived from livers and hearts of mice depleted Lats1/2. o, AAV-mediated knockdown of Lats1/2 increases liver size. Data were shown as mean ± s.e.m. (n = 5 mice/group). P values in f, g, j and o were calculated using two-tailed Student’s t-test. The experiments in b, l, m and n were repeated n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 9. Statistical source data are available in Statistical Source Data Extended Data Fig. 9.

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Supplementary information

Reporting Summary

Supplementary Table 1

Primers for RT–PCR and siLATS1/2

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Gan, W., Dai, X., Dai, X. et al. LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control. Nat Cell Biol 22, 246–256 (2020). https://doi.org/10.1038/s41556-020-0463-6

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