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The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1

Nature Cell Biology volume 24, pages 74–87 (2022)Cite this article

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Abstract

Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.

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Fig. 1: Hippo signalling deficiency protects cells against heavy metal-induced toxicity.
Fig. 2: Hippo signalling deficiency induces expression of heavy metal response genes.
Fig. 3: The Hippo pathway controls the heavy metal response by inhibiting the transcriptional activity of MTF1.
Fig. 4: LATS1 binds and phosphorylates MTF1 at S152.
Fig. 5: LATS-mediated MTF1 S152 phosphorylation inhibits the heavy metal response.
Fig. 6: Inhibition of the Hippo pathway promotes heavy metal detoxification.
Fig. 7: The Hippo pathway is negatively regulated by heavy metals.
Fig. 8: Zn binds and inhibits LATS.

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Data availability

The RNAseq dataset is available in the Gene Expression Omnibus under the accession number GSE163156. The lung squamous cell carcinoma RNAseq datasets were downloaded from The Cancer Genome Atlas data portal (https://portal.gdc.cancer.gov/). The protein structure information (PDB: 1ZW8) was downloaded from the Protein Data Bank (https://www.rcsb.org/structure/1zw8). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank K.-L. Guan (University of California, San Diego), D. Pan (The University of Texas Southwestern Medical Center), D. Fruman (University of California, Irvine) and A. Edinger (University of California, Irvine) for comments on this study. We thank J. W. C. Leung (University of Arkansas for Medical Sciences), R. Edwards (University of California, Irvine), G. MacGregor (University of California, Irvine) and R. Geertsema (University of California, Irvine) for their technical help. This work was supported by NIH grants (grant nos R01GM126048 and R21ES031642), an American Cancer Society Research Scholar grant (grant no. RSG-18-009-01-CCG), a University of California Cancer Research Coordinating Committee Research Award (grant no. CRN-19-585568), and a Tower Cancer Research Foundation Cancer Free Generation Career Development Grant to W.W.; and was also supported in part by NIH grants to R.L. (grant no. R35GM130367), K.W.Y.C. (grant no. R35GM139617) and L.H. (grant no. R01GM130144). W.W. is a member of the Chao Family Comprehensive Cancer Center (award no. P30CA062203) at the University of California, Irvine.

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Authors and Affiliations

  1. Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA

    Han Han, Hiroki J. Nakaoka, Line Hofmann, Jeff Jiajing Zhou, Gayoung Seo, Rebecca Elizabeth Vargas, Bing Yang, Lee Bardwell, Ken W. Y. Cho, Rahul Warrior & Wenqi Wang

  2. Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA

    Clinton Yu & Lan Huang

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA

    Lisha Zeng, Junyu Nan & Ray Luo

  4. Cryo-EM Center, Southern University of Science and Technology, Shenzhen, China

    Ruxi Qi

  5. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    Dmitry A. Fishman

  6. Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA

    Ray Luo

  7. Department of Materials Science and Engineering, University of California, Irvine, Irvine, CA, USA

    Ray Luo

  8. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA

    Ray Luo

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  1. Han Han
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Contributions

H.H. and W.W. conceived and designed the study. W.W. supervised the study. H.H. and H.J.N. performed all of the experiments with the assistance from G.S., R.E.V. and B.Y. L.H. and R.W. performed the Drosophila survival study. C.Y. and L.H. performed the mass spectrometry analysis. L.B. helped with the in vitro kinase assay. J.N., L.Z., R.Q. and R.L. performed the simulation analysis. J.J.Z. and K.W.Y.C. helped with the RNAseq analysis. D.A.F. helped with the circular dichroism analysis. W.W. wrote the manuscript.

Corresponding authors

Correspondence to Rahul Warrior or Wenqi Wang.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Inhibition of the Hippo pathway activates heavy metal response.

(a) Validation of the YAP-5SA overexpressed HEK239A cells. Western blot was performed using the indicated antibodies. (b) Validation of the YAP/TAZ shRNAs-transduced LATS1/2 DKO HEK293A cells. Western blot was performed using the indicated antibodies. (c,d) Western blot was performed using the indicated antibodies (c). MT1A gene transcription was examined in the indicated cells treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours by qPCR (mean ± s.d., n = 3 biological replicates) (d). *** p < 0.001 (two-tailed Student’s t-test). Relative expression fold change numbers are shown. (e,f) Transcription of MT1F (e) and CTGF (f) was examined in the HEK293A cells cultured under serum starvation and serum stimulation by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). (g,h) Transcription of MT1F (g) and CTGF (h) was examined in the HEK293A cells cultured under high density and low density by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). (i,j) Serum-starved wild-type and the LATS1/2 DKO HEK293A cells were treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. Transcription of MT1A (i) and MT1F (j) was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). Relative expression fold change numbers are shown. (k-l) Wild-type and the LATS1/2 DKO HEK293A cells were treated with 250 μM Zn with the indicated anions for 4 hours. The transcription of MT1A (i) and MT1F (j) was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). (m) Validation of the indicated HEK293A cells stably expressing SFB-MTF1. Western blot was performed using the indicated antibodies. Data shown represent 2 independent experiments in a-c, m.

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Extended Data Fig. 2 LATS1 binds and phosphorylates MTF1.

(a) HEK293A and Hep3B cells stably expressing SFB-tagged MTF1 were subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. (b) HEK293A cells were transfected with the construct encoding SFB-tagged MTF1, treated with CdCl2 (50 µM) and ZnCl2 (250 µM) for 1 hour, and subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. (c) SFB-tagged LATS1 and its kinase dead (K734R) mutant were expressed in HEK293T cells, purified using S protein beads, and subjected to the ATP-γ-S-based in vitro kinase assay. Bacterially purified MBP-MTF1-R2, MBP-R2-5A mutant (S152A/T153A/T241A/S301A/T302A) and GST-YAP proteins were used as substrates. Western blot was performed using the indicated antibodies. (d) HEK293A cells stably expressing SFB-tagged MTF1 were transfected with the constructs encoding HA-tagged LATS1 and its kinase dead (K734R) mutant, and subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. Arrow indicates the exogenously expressed SFB-MTF1. Pound sign indicates endogenous MTF1. (e) SFB-tagged LATS1, STK38 and STK38L were expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay. Bacterially purified MBP-tagged MTF1-R2 protein was used as substrate. Western blot was performed using the indicated antibodies. Data shown represent 2 independent experiments in a-e.

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Extended Data Fig. 3 MTF1 S152 phosphorylation inhibits heavy metal response.

(a,b) The indicated HEK293A cells were treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. The transcription of MT1A (a) and MT1F (b) was examined by qPCR (mean ± s.d., n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). ns, no significance. Relative expression fold change numbers are shown. (c) Validation of the MTF1 KO HEK293A cells reconstituted with the indicated SFB-tagged MTF1 and its mutants. Western blot was performed using the indicated antibodies. Arrow indicates the exogenously expressed SFB-tagged MTF1 and its mutants. (d,e) The MTF1KO HEK293A cells were reconstituted with the indicated SFB-tagged MTF1 and its mutants, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 12 hours, visualized by crystal violet staining (d), and quantified for relative viability (mean ± s.d., n = 4 biological replicates) (e). *** p < 0.001 (two-tailed Student’s t-test). (f,g) The transcription of MT1A (f) and MT1F (g) was examined in wild-type and the MTF1 KO HEK293A cells reconstituted with the indicated SFB-tagged MTF1 and its mutants by qPCR (mean ± s.d., n = 3 biological replicates). Cells were treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). ns, no significance. (h) The structure comparison of the ZAP1-ZNF1 in khaki with the phosphorylated ZAP1-ZNF1 (p-S591) in pink (left) and with ZAP1-ZNF1 S591D mutant in blue (right). The overall RMSD values of wild-type ZAP1-ZNF1 with its phosphorylated form (p-S591) and S591D mutant are shown. Arrows indicate the S591 site. (i) The MTF1 KO HEK293A cells were reconstituted with the indicated SFB-MTF1 and its mutants, and treated with ZnCl2 (250 μM) for 1 hour. Immunofluorescent staining was performed. Scale bar, 40 μm. Data shown represent 3 independent experiments. (j) The MTF1 KO HEK293A cells were reconstituted with the indicated SFB-MTF1 and its mutants, treated with ZnCl2 (250 μM) for 1 hour, and subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. Data shown represent 2 independent experiments in c, j.

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Extended Data Fig. 4 Characterization of XMU-MP-1 in regulating the Hippo pathway and heavy metal response.

(a) Immunohistochemical analyses of Mtf1 and its S151 phosphorylation were performed in the liver and kidney tissues of the mice treated with DMSO, XMU-MP-1 alone or combined with VP post CdCl2 intoxication. The indicated regions in the box are shown 3 times enlarged. Scale bar, 150 μm. Data shown represent 3 independent experiments. (b) Wild-type and the MST1/2 DKO Hep3B and HEK293A cells were treated with DMSO and XMU-MP-1 (10 μM) for 4 hours, and subjected to Western blot analysis using the indicated antibodies. (c,d) The indicated Hep3B cells (c) and HEK293A cells (d) were pre-incubated with XMU-MP-1 (10 μM) for 2 hours, and treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. The transcription of MT1F was examined by qPCR (mean ± s.d., n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). ns, no significance. (e-h) Hep3B and HEK293A cells stably expressing SFB-tagged TAOK1 (e), MAP4K2 (f), MAP4K4 (g) and MAP4K6 (h) were serum starved for 24 hours, and treated with XMU-MP-1 (10 μM) for 4 hours. The indicated SFB-tagged kinases were purified using S protein beads and subjected to in vitro kinase assay using the bacterially purified MBP-tagged LATS1-C3 protein as substrate. Western blot was performed using the indicated antibodies Data shown represent 2 independent experiments in b, e-h.

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Extended Data Fig. 5 The Hippo pathway-mediated regulation of MTF1 and heavy metal response is conserved in Drosophila.

(a) Heterozygous mats Drosophila mutant and homozygous mtf1 Drosophila mutant were used for adult viability assay. Adult viability was assayed by feeding adult Drosophila (~7-day old) with normal food supplemented with 5 mM CuSO4 and determining the number of survivors daily. **, p < 0.01; ***, p < 0.001 (Log-rank test). n = 19 for wild-type Drosophila, n = 14 for mtf-/- Drosophila, n = 24 for mats−/+ Drosophila. (b) The MTF1 KO HEK293A cells were transfected with the constructs encoding the SFB-tagged human MTF1 and fly-mtf1. Western blot was performed using the indicated antibodies. (c) The MTF1 KO HEK293A cells were transfected with the constructs encoding the SFB-tagged human MTF1 and fly-mtf1, and treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. MT1A gene transcription was examined by qPCR (mean ± s.d., n = 3 biological replicates). *** p < 0.001 (two-tailed Student’s t-test). (d) HEK293A cells were transfected with the constructs encoding the SFB-tagged fly-mtf1 and its S126D mutant, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 1 hour, and subjected to immunofluorescent staining. Scale bar, 40 μm (e) The LATS/MTF1 TKO HEK293A cells were transduced with the constructs encoding the SFB-tagged fly-mtf1 and its S126D mutant. Western blot was performed using the indicated antibodies. (f) The LATS/MTF1 TKO HEK293A cells stably expressing the SFB-tagged fly-mtf1 and its S126D mutant were treated with CdCl2 (50 μM), ZnCl2 (250 μM) and CuSO4 (250 μM) for 4 hours. MT1F gene transcription was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). Data shown represent 2 independent experiments in b, d, e.

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Extended Data Fig. 6 Heavy metals inhibit the Hippo pathway in different cells and tissues.

(a) Hep3B cells were serum starved for 24 hours, and treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for the indicated time points. Western blot was performed using the indicated antibodies. (b) MCF10A and HeLa cells were serum starved for 24 hours, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 1 hour, and subjected to immunofluorescent staining. Scale bar, 40 μm (c) MCF10A and HeLa cells were serum starved for 24 hours, and treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 4 hours. The transcription of YAP downstream genes CTGF and CYR61 was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). (d) Hep3B cells stably expressing SFB-tagged MTF1 were serum starved for 24 hours, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for the indicated time points, and subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. (e) Hep3B and HEK293A cells were serum starved for 24 hours, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 1 hour, and subjected to immunoprecipitation using LATS1 antibody. Western blot was performed using the indicated antibodies. Arrow indicates the correct p-LATS1 S909 signal. Asterisk indicates the non-specific signal. (f) The transcription of heavy metal response gene Mt1 was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). Relative expression fold change numbers are shown. (g) The transcription of Yap downstream gene Ctgf was examined by qPCR (mean ± s.d., n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). Relative expression fold change numbers are shown. (h-j) The Human Protein Atlas (https://www.proteinatlas.org/) was used to examine the expression of MTF1 and LATS1 in different human tissues (h), human cells (i) and commonly used human cell lines (j). Data shown represent 2 independent experiments in a, b, d, e.

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Extended Data Fig. 7 Zn binds and inhibits LATS.

(a) Illustration of the metal beads. (b) HEK293T cells were transfected with the constructs encoding the indicated Hippo pathway components and subjected to pulldown assay using Cu beads. The input blot is shared with the one in Fig. 8c. (c,d) HEK293T cells were transfected with the constructs encoding SFB-tagged LATS1 (c) and LATS2 (d), and subjected to pulldown assay using Zn beads in the presence of TPEN at the indicated concentrations. (e-f) SFB-tagged MST1 was expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay in the presence of CaCl2 (e) and ZnCl2 (f) at the indicated concentrations. MBP-tagged LATS1-C3 protein was used as substrate. (g) SFB-tagged MAP4K2 was expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay in the presence of ZnCl2 at the indicated concentrations. MBP-tagged LATS1-C3 protein was used as substrate. (h) SFB-tagged MST1 was expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay in the presence of ZnCl2 at the indicated concentrations. GST-tagged MOB1 protein was used as substrate. (i-j) HEK293A cells were transfected with the constructs encoding the SFB-tagged LATS1 with HA-tagged PPP1CA (i) and POPX2 (j), treated with ZnCl2 (250 μM) and CaCl2 (250 μM) for 1 hour, and subjected to pulldown assay using S protein beads. (k-l) SFB-tagged LATS1 was expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay in the presence of CaCl2 (k) or ZnCl2 (l) at the indicated concentrations. GST-tagged YAP protein was used as substrate. (m) SFB-tagged LATS2 was expressed in HEK293T cells, purified using S protein beads, and subjected to in vitro kinase assay in the presence of ZnCl2 at the indicated concentrations. GST-tagged YAP protein was used as substrate. (n) The potential Zn-binding Cys, His, Glu and Asp residues in the LATS1-C3 region are shown. The conserved Cys and His residues are highlighted in yellow. The conserved Glu and Asp residues are highlighted in blue. LATS1 T1079 site is indicated by arrow. Data shown represent 2 independent experiments in b-m.

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Extended Data Fig. 8 The Hippo pathway mediates cellular response to different heavy metals.

(a) HEK293A cells were transfected with the construct encoding SFB-tagged MOB1, treated with CdCl2 (50 μM) and ZnCl2 (250 μM) for 1 hour, and subjected to pulldown assay using S protein beads. Western blot was performed using the indicated antibodies. short, short exposure; long, long exposure. (b) HEK293T cells were transfected with the construct encoding SFB-tagged MOB1 and subjected to pulldown assay using the indicated metal beads. (c) Hep3B and HEK293A cells were serum starved for 24 hours and treated with CuSO4 (250 μM) and ZnCl2 (250 μM) for 4 hours. The transcription of MT1A, MT1F and CYR61 was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). (d) The indicated cells were treated with CuSO4 (250 μM) and ZnCl2 (250 μM) for 4 hours. The transcription of MT1A was examined by qPCR (mean ± s.d., n = 3 biological replicates). *** p < 0.001 (two-tailed Student’s t-test). (e) SFB-tagged LATS1 was expressed in HEK293T cells, purified using S protein beads, washed thoroughly with high-salt buffer containing 250 mM NaCl, incubated with Zn chelator TPEN (25 μM) or Cu chelator TTM (25 μM), and subjected to in vitro kinase assay. GST-YAP protein used as substrate. (f) Illustration of cisplatin and carboplatin compound structures, where platinum is labelled in red. (g) The indicated cells were treated with cisplatin (10 μM) and carboplatin (100 μM) for 6 hours. The transcription of MT1A was examined by qPCR (mean ± s.d., n = 3 biological replicates). ** p < 0.05, *** p < 0.001 (two-tailed Student’s t-test). ns, no significance. Relative expression fold change numbers are shown. (h-k) The lung squamous cell carcinoma (LUSC) RNAseq datasets with clinic data were downloaded from the Cancer Genome Atlas (TCGA) data portal. The indicated gene expression from a total of 119 patients treated with cisplatin or carboplatin were subjected to Spearman correction analysis. Correlation coefficient R value and p value were calculated by GraphPad prism software. (l) A proposed model for the Hippo pathway-mediated heavy metal response through MTF1. Data shown represent 2 independent experiments in a, b, e.

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Supplementary information

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Supplementary Table 1

Supplementary Table 1. A total of 181 LATS1/2-regulated heavy metal response genes are listed based on clustered groups as shown in Fig. 2c. Supplementary Table 2. Sequence information of the qPCR primers used in this study.

Supplementary Video 1

The H2O control-treated mice are shown (upper left). Mice were pretreated with CdCl2 for 1 h and subjected to treatment with vehicle (upper right), XMU-MP-1 (lower left) or XMU-MP-1 + verteporfin (lower right). This video was taken 4 h post the CdCl2 intoxication.

Supplementary Video 2

The H2O control-treated mice are shown (upper left). Mice were pretreated with CdCl2 for 1 h and subjected to treatment with vehicle (upper right), XMU-MP-1 (lower left) and XMU-MP-1 + verteporfin (lower right). This video was taken 100 h post the CdCl2 intoxication.

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Han, H., Nakaoka, H.J., Hofmann, L. et al. The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1. Nat Cell Biol 24, 74–87 (2022). https://doi.org/10.1038/s41556-021-00813-8

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