AMPK modulates Hippo pathway activity to regulate energy homeostasis

Abstract

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.

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Figure 1: Glucose homeostasis controls YAP phosphorylation and localization.
Figure 2: Release from energy stress activates YAP.
Figure 3: AMPK associates with and phosphorylates YAP.
Figure 4: AMPK phosphorylates YAP at S61 and suppresses its transcriptional activity.
Figure 5: Energy stress increases LATS kinase activation independent of MST kinases.
Figure 6: YAP regulates GLUT3 transcription in the glucose metabolic pathway.
Figure 7: YAP and GLUT3 expression positively correlate with each other in human colon and liver cancers.

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Acknowledgements

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).

Author information

W.W. performed all of the experiments with assistance from Z-D.X., X.L., K.E.A., B.G., R.L.J. and J.C. W.W. and J.C. designed the experiments. J.C. supervised the study. W.W. and J.C. wrote the manuscript. All authors commented on the manuscript.

Correspondence to Junjie Chen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Identification of AMPK phosphorylation sites on YAP by mass spectrometry.

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.

Supplementary Figure 3 Validation of YAP S61 phospho-antibody.

(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. (CD) 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.

Supplementary Figure 4 AMPK is not essential for Hippo pathway activation under energy stress.

(AB) 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. (BC) 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.

Supplementary Figure 6 Predicted TEAD-binding site in the GLUT3 promoter region.

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.

Supplementary Table 1 Identification of YAP phosphorylation site by AMPK

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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

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