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Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway

Nature Cell Biology volume 17, pages 500–510 (2015)Cite this article

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Abstract

YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size. YAP is inhibited by the kinase Lats, which phosphorylates YAP to induce its cytoplasmic localization and proteasomal degradation. YAP induces gene expression by binding to the TEAD family transcription factors. Dysregulation of the Hippo–YAP pathway is frequently observed in human cancers. Here we show that cellular energy stress induces YAP phosphorylation, in part due to AMPK-dependent Lats activation, thereby inhibiting YAP activity. Moreover, AMPK directly phosphorylates YAP Ser 94, a residue essential for the interaction with TEAD, thus disrupting the YAP–TEAD interaction. AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats-null cells with high YAP activity. Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo–YAP pathway.

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Figure 1: Cellular energy starvation activates Lats and inhibits YAP.
Figure 2: AMPK is required for Hippo–YAP regulation by energy stress.
Figure 3: Energy stress induces YAP phosphorylation via both Lats-dependent and Lats-independent mechanisms.
Figure 4: AMPK phosphorylates Ser 94 in YAP.
Figure 5: AMPK inhibits YAP activity through phosphorylation of Ser 94.
Figure 6: Energy stress attenuates tumorigenicity of Lats1/2 DKO MEFs.
Figure 7: AMPK is required for energy stress to inhibit YAP activity.

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  • 14 April 2015

    In the Methods section 'Antibodies and reagents' there was a typographical error in the company name GeneTex. The sentence should have read 'Anti-phosphorylated Ser 94 antibody was generated by immunizing rabbits with phosphopeptides (Abbiotec and GeneTex, 1:500)'. This has now been corrected online.

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Acknowledgements

We would like to thank A. Hong and F. Flores for critical reading of the manuscript. This work was supported by grants from National Institutes of Health (CA132809, EY022611, DEO15964 and CA23100, K-L.G.). C.G.H. is supported by a Postdoctoral Fellowship from the Danish Council for Independent Research, Natural Sciences.

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Authors and Affiliations

  1. Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA

    Jung-Soon Mo, Zhipeng Meng, Hyun Woo Park, Carsten Gram Hansen, Soohyun Kim & Kun-Liang Guan

  2. Department of Cardiology, Veterans Medical Research Foundation, 3350 La Jolla Village Dr., San Diego, California 92161, USA

    Young Chul Kim

  3. Department of Biological Sciences, National Creative Research Initiatives Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea

    Dae-Sik Lim

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Contributions

J-S.M. and K-L.G. designed the experiments, analysed data and wrote the paper. J-S.M. performed the experiments with assistance from Z.M., Y.C.K., H.W.P., C.G.H. and S.K.; D-S.L. established the Lats knockout MEFs. All authors discussed the results and commented on the manuscript.

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Correspondence to Kun-Liang Guan.

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Integrated supplementary information

Supplementary Figure 1 Energy stress induces YAP phosphorylation.

(a) Osmotic stress does not affect YAP and TAZ mobility shift. HEK293A cells were treated with different concentrations of 2-DG or sorbitol for 1 h. Cells were then lysed and subjected to immunoblotting using the indicated antibodies. (b) 2-DG induces YAP phosphorylation. HEK293A cells were treated with different concentrations of 2-DG for 30 min. Cells were then lysed and cell lysates were subjected to immunoblotting using the indicated antibodies. (c) Experiments are the same as in panel an exception cells were treated with 25 mM 2-DG for various times. (d–f) Effect of 2-DG on YAP phosphorylation in different cell lines. C2C12 cells were treated with 10 mM and 25 mM 2-DG (d); MCF10A cells were treated with 10 and 25 mM 2-DG (e); HeLa cells were treated with 10 and 25 mM (f). (g) Glucose starvation inhibits YAP and TEAD1 interaction. HEK293A cells were starved with glucose for 2 h as indicated. Endogenous YAP/TAZ and the co-immunoprecipitated TEAD were detected by western blot.

Supplementary Figure 2 2-DG does not affect MST phosphorylation.

(a) Effect of MST overexpression on 2-DG induced YAP phosphorylation. HEK293A cells were transiently co-transfected with indicated plasmids. After transfection, cells were treated with 25 mM 2-DG for 1 h and YAP phosphorylation status was determined by phos-tag gel. (b) 2-DG does not affect MST1 phosphorylation. Endogenous MST1 was immunoprecipitated from control or 2-DG (25 mM, 2 h) treated HEK293A cells. MST1 phosphorylation was detected by a phospho-MST (T183) specific antibody.

Supplementary Figure 3 Activation of AMPK increases YAP phosphorylation in various cell lines.

(a) Genotyping of AMPKa1 and AMPKa2 wild-type (WT) and mutant (KO) alleles. DNA was isolated from AMPK+/+ or AMPK−/− cells as indicated. Genotyping was performed by PCR using primers specific to wild-type AMPKa (left side of the dash line) or AMPKa KO (right side of the dash line). The PCR products were run on the same agarose gel. Samples in the left panel are PCR products using primer specific to AMPKa1 where samples in the right panel are PCR products using primers specific to AMPKa2. (b) Cells were treated with 25 mM 2-DG for various times as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated for 1 h. Endogenous YAP mobility was examined by western blot. (c) AICAR increases YAP phosphorylation in hepatocytes. Primary mouse hepatocytes were treated with different doses of AICAR for 8 h. Cell lysates were used for immunoblotting with indicated antibodies. (d) HepG2 cells were treated with different doses of AICAR for 2 h. (e) HepG2 cells were treated with different doses of A769662 for 2 h.

Supplementary Figure 4 Identification of AMPK phosphorylation sites in YAP.

(a) AMPK induces mobility shift of YAP-5SA. GST-YAP 5SA was used as substrates for in vitro AMPK phosphorylation. GST- YAP mobility was examined by western blot (b) Mass spectrometry analyses of AMPK-phosphorylated YAP. GST-YAP-5SA was purified from E.coli and was used as a substrate for an in vitro AMPK assay. After electrophoresis, the phosphorylated GST-YAP 5SA band was cut and digested with trypsin/thermolysine followed by mass spectrometry analyses. The green coloured bars (top) and amino acid residues (lower part) are sequences that were detected by mass spectrometry. Phosphorylation sites identified by mass spectrometry are indicated on top of the amino acid sequence. (c) S94 is the major AMPK phosphorylation site in the YAP (51-121) fragment. Experiments were similar to panel a. (d) All three indicated residues of YAP were mutated to alanine. A luciferase reporter controlled by multiple TEAD binding sequences was transfected into HEK293T cells together with 5 × UAS-luciferase reporter for Gal4-TEAD4 and Renilla constructs and indicated plasmids. After 48 h, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control. (d) Two different substrates of AMPK, TSC2 and ULK1, and YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP Phosphorylation was detected by autoradiography. GST-ULK1 (279-425), TSC2 (1300-1367) and YAP fragment proteins were detected by Coomassie staining. (e) Experiments are same as in panel c. except that a GST-YAP truncated form and UKL1 were used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP for the indicated times. (f) Experiments are the same as in panel d. exception GST-YAP WT and UKL1 used as substrate for the indicated times. (g) AMPK has no effect on the YAP and RUNX2 interaction. HEK293A cells were transfected with the indicated plasmids. Flag-RUNX2 was immunoprecipitated and the co-precipitated HA-YAP was detected by Western blot.

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Mo, JS., Meng, Z., Kim, Y. et al. Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Nat Cell Biol 17, 500–510 (2015). https://doi.org/10.1038/ncb3111

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