The Hippo pathway was discovered as a conserved tumour suppressor pathway restricting cell proliferation and apoptosis. However, the upstream signals that regulate the Hippo pathway in the context of organ size control and cancer prevention are largely unknown. Here, we report that glucose, the ubiquitous energy source used for ATP generation, regulates the Hippo pathway downstream effector YAP. We show that both the Hippo pathway and AMP-activated protein kinase (AMPK) were activated during glucose starvation, resulting in phosphorylation of YAP and contributing to its inactivation. We also identified glucose-transporter 3 (GLUT3) as a YAP-regulated gene involved in glucose metabolism. Together, these results demonstrate that glucose-mediated energy homeostasis is an upstream event involved in regulation of the Hippo pathway and, potentially, an oncogenic function of YAP in promoting glycolysis, thereby providing an exciting link between glucose metabolism and the Hippo pathway in tissue maintenance and cancer prevention.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Pan, D. The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 (2010).
Zhao, B., Li, L., Lei, Q. & Guan, K. L. The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. Genes Dev. 24, 862–874 (2010).
Halder, G. & Johnson, R. L. Hippo signaling: growth control and beyond. Development 138, 9–22 (2011).
Zhao, B., Li, L., Tumaneng, K., Wang, C. Y. & Guan, K. L. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ−TRCP. Genes Dev. 24, 72–85 (2010).
Jiao, S. et al. A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer. Cancer Cell 25, 166–180 (2014).
Zhang, W. et al. VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex. Cell Res. 24, 331–343 (2014).
Lu, L. et al. Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver. Proc. Natl Acad. Sci. USA 107, 1437–1442 (2010).
Cai, J. et al. The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev. 24, 2383–2388 (2010).
Lee, K. P. et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc. Natl Acad. Sci. USA 107, 8248–8253 (2010).
Camargo, F. D. et al. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr. Biol. 17, 2054–2060 (2007).
Dong, J. et al. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130, 1120–1133 (2007).
Mo, J. S., Park, H. W. & Guan, K. L. The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 15, 642–656 (2014).
Zhao, B. et al. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747–2761 (2007).
Zhao, B. et al. Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 26, 54–68 (2012).
Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13, 591–600 (2012).
Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).
Yu, F. X. et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150, 780–791 (2012).
Jones, R. G. & Thompson, C. B. Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev. 23, 537–548 (2009).
Cairns, R. A., Harris, I. S. & Mak, T. W. Regulation of cancer cell metabolism. Nat. Rev. Cancer 11, 85–95 (2011).
Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461 (2011).
Crute, B. E., Seefeld, K., Gamble, J., Kemp, B. E. & Witters, L. A. Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase. J. Biol. Chem. 273, 35347–35354 (1998).
Hao, Y., Chun, A., Cheung, K., Rashidi, B. & Yang, X. Tumor suppressor LATS1 is a negative regulator of oncogene YAP. J. Biol. Chem. 283, 5496–5509 (2008).
Fujishiro, S. H. et al. ERK1/2 phosphorylate GEF-H1 to enhance its guanine nucleotide exchange activity toward RhoA. Biochem. Biophys. Res. Commun. 368, 162–167 (2008).
Wu, S. Z. et al. Akt and RhoA activation in response to high glucose require caveolin-1 phosphorylation in mesangial cells. Am. J. Physiol. Renal Physiol. 306, F1308–F1317 (2014).
Zhang, Y., Peng, F., Gao, B., Ingram, A. J. & Krepinsky, J. C. High glucose-induced RhoA activation requires caveolae and PKCbeta1-mediated ROS generation. Am. J. Physiol. Renal Physiol. 302, F159–F172 (2012).
Zhao, B. et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22, 1962–1971 (2008).
Stubbs, M., McSheehy, P. M., Griffiths, J. R. & Bashford, C. L. Causes and consequences of tumour acidity and implications for treatment. Mol. Med. Today 6, 15–19 (2000).
Zhang, H., Pasolli, H. A. & Fuchs, E. Yes-associated protein (YAP) transcriptional coactivator functions in balancing growth and differentiation in skin. Proc. Natl Acad. Sci. USA 108, 2270–2275 (2011).
Yamamoto, T. et al. Over-expression of facilitative glucose transporter genes in human cancer. Biochem. Biophys. Res. Commun. 170, 223–230 (1990).
Mellanen, P., Minn, H., Grenman, R. & Harkonen, P. Expression of glucose transporters in head-and-neck tumors. Int. J. Cancer 56, 622–629 (1994).
Boado, R. J., Black, K. L. & Pardridge, W. M. Gene expression of GLUT3 and GLUT1 glucose transporters in human brain tumors. Brain Res. Mol. Brain Res. 27, 51–57 (1994).
Younes, M., Brown, R. W., Stephenson, M., Gondo, M. & Cagle, P. T. Overexpression of Glut1 and Glut3 in stage I nonsmall cell lung carcinoma is associated with poor survival. Cancer 80, 1046–1051 (1997).
Ayala, F. R. et al. GLUT1 and GLUT3 as potential prognostic markers for oral squamous cell carcinoma. Molecules 15, 2374–2387 (2010).
Ha, T. K. & Chi, S. G. CAV1/caveolin 1 enhances aerobic glycolysis in colon cancer cells via activation of SLC2A3/GLUT3 transcription. Autophagy 8, 1684–1685 (2012).
Mo, J-S. et al. Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Nat. Cell Biol. 17, http://dx.doi.org/10.1038/ncb3111 (2015).
DeRan, M. et al. Energy stress regulates Hippo-YAP signaling involving AMPK-mediated regulation of angiomotin-like 1 protein. Cell Rep. 9, 495–503 (2014).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).
Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001).
Eng, J. K., McCormack, A. L. & Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).
Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).
We thank all of our colleagues in the Chen laboratory for insightful discussion and technical assistance, especially J. Yuan, G. Ghosal and B. C. Nair. We also thank J-I. Park, L. Ma, Y. Sun, J. Zhang, A. Lin and H. Lee for reagents, insightful suggestions and comments on this work. We thank F-X. Yu (University of California, San Diego) for technical help. We thank the shRNA-ORFeome Core Facility at MD Anderson Cancer Center for the ORFs and shRNAs. We thank B. Viollet for providing AMPK wild-type and knockout MEFs. We thank K. Hale for proof-reading the manuscript. We thank R. Tomaino for assistance with the mass spectrometry analysis. This work was supported in part by the US Department of Defense Era of Hope research scholar award to J.C. (W81XWH-09-1-0409 and W81XWH-05-1-0470). This work was partly supported by the U.S. National Cancer Institute through the MD Anderson Cancer Center Support Grant (CA016672).
The authors declare no competing financial interests.
Integrated supplementary information
Bacteria-purified GST-YAP (2 μg) was used as substrate for AMPK in vitro kinase assay in the presence of cold ATP (500 μM) as described in the Supplementary Methods. Phosphorylation analysis of YAP was performed by the Taplin Mass Spectrometry Facility. Information on the identified phospho-peptides is shown.
(A) 2-DG induced YAP 5SA mutant phosphorylation. HEK293A cells were transfected with indicated plasmids and treated with glucose-free medium supplemented with 2-DG for 4 hours. Cell lysates were subjected to western blotting. (B) In vitro kinase assay using purified AMPK kinase. SFB-AMPKα1 or its kinase-dead mutant (K47R) was co-expressed with Flag-AMPKβ2 or Flag-AMPKγ2 plasmid in 293T cells for 48 hours. AMPK complex was purified by S protein beads and subjected to the in vitrokinase assay, where GST-ACC (residues 1-130) (2 μg) was purified from bacterial and taken as the substrate. Reaction samples were subjected to western blotting with indicated antibodies to verify the AMPK-dependent phosphorylation of ACC. (C–D) Validation of YAP S61 phospho-antibody. YAP S61 phospho-antibody was used to detect AMPK-mediated YAP phosphorylation by the in vitro kinase assay (C). Short exp and long exp represent short exposure and long exposure respectively. YAP S61 antibody was also used to detect AMPK active truncation mutant-mediated YAP phosphorylation in vivo (D). AMPK kinase-dead mutant (K47R) and active AMPK truncation dead mutant (T172A) were used as negative controls for AMPK. YAP-S61A mutant was used as the control for wild-type YAP. (E) Sequence alignment of the AMPK phosphorylation sites in YAP from different species and other known AMPK substrates. Uncropped images of western blots are shown in Supplementary Fig. 7.
(A–B) AMPK did not noticeably affect LATS1 and MST1 activation. SFB tagged LATS1 (A) or MST1 (B) was co-expressed with active AMPK truncation mutant in 293T cells. Cell lysates were subjected to western blotting with indicated antibodies. (C) LATS1 kinase activation was observed in both wild-type and AMPK-deficient cells. Wild-type MEFs (WT) and AMPKα-knockout MEFs (AMPKα KO) were treated with 2-DG (25 mM) in glucose-free medium and rapamycin (50 nM) for 4 hours. Western blotting analysis was performed using indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. 7.
Supplementary Figure 5 AMPK-regulated YAP phosphorylation does not affect 14-3-3 binding or subcellular localization of YAP, but suppresses its transcriptional activity.
(A) Phosphorylation of YAP by AMPK did not affect the association of YAP with the 14-3-3 protein. Indicated plasmids were co-expressed in 293T cells. Cell lysates were used for pulldown assay with S protein beads and subjected to western blotting. (B–C) Phosphorylation of YAP on the S61 site was not required for the binding of YAP to 14-3-3 (B). The active AMPK truncation mutant did not increase the association between the YAP-4SA-61S mutant and 14-3-3 (C). (D) AMPK did not affect cellular localisation of YAP. HeLa cells with exogenously expressed active AMPK truncation were subjected to immunostaining with the GST and YAP antibodies. Nuclei were visualized by Dapi. Scale bar = 20μm. (E) S61 mutation did not affect YAP localisation. HeLa cells with exogenously expressed indicated YAP mutants were subjected to immunostaining with Flag antibody. Nuclei were visualised by Dapi. Scale bar = 20μm. (F) Phospho-mimetic mutation of the S94 site (S94D), but not the 61 site (S61D), interfered with YAP-TEAD binding. Indicated plasmids were co-expressed in 293T cells. Cells lysates were used for pulldown assay with S protein beads and subjected to western blotting. (G) Phospho-mimetic mutants of the S61 site (S61D) and the S94 site (S94D) suppressed the transcription of YAP downstream genes. The transcripts of YAP-regulated genes (CTGF and CYR61) were detected by quantitative PCR in indicated YAP stable cells and normalised (mean ± s.d, n = 3 biological replicates). ∗∗∗P < 0.001(Student t-test). Statistics source data are shown in Supplementary Table 2. Uncropped images of western blots are shown in Supplementary Fig. 7.
A conserved TEAD binding site was identified in the GLUT3 promoter region. ENCODE data showed that this site was occupied by TEAD transcription factor.
Supplementary Figure 7 A proposed model of YAP regulation by AMPK and Hippo pathway under energy stress.
YAP is phosphorylated and suppressed in response to energy stress. Both LATS kinase and AMPK are activated by energy stress and contribute to YAP inhibition via phosphorylating it on multiple sites. Energy stress induced-LATS kinase activation largely depends on the suppression of Rho GTPase and actin cytoskeleton dynamics, but it is also indirectly regulated by AMPK. With glucose is abundant, YAP translocates into nucleus and promotes glycolysis at least in part by upregulating GLUT3 at transcriptional level.
About this article
Cite this article
Wang, W., Xiao, Z., Li, X. et al. AMPK modulates Hippo pathway activity to regulate energy homeostasis. Nat Cell Biol 17, 490–499 (2015). https://doi.org/10.1038/ncb3113
Biological and Pharmaceutical Bulletin (2020)
The creatine–phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis
Nature Metabolism (2020)
Nature Reviews Drug Discovery (2019)
Frontiers in Bioengineering and Biotechnology (2019)
Journal of Cardiovascular Pharmacology and Therapeutics (2019)