Abstract
The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ1,2. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD)3. The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components2. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.
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Acknowledgements
This work was supported by grants from the National Institutes of Health (CA196878, DE15964 and GM51586) to K.-L.G., the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A5A2009936) to H.-S.J. and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C1560), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MOE) (2017R1D1A1B03034797) and (MSIP) (2017R1A4A1015328) to H.W.P. K.C.L. and S.W.P. were supported in part by the University of California, San Diego (UCSD) Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).
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K.C.L. and H.W.P. designed experiments, performed research, analysed data and wrote the manuscript. T.M., Z.M., H.-S.J. and S.W.P. designed experiments, performed research, analysed data and reviewed the manuscript. Y.S. and J.H. provided reagents and reviewed the manuscript. K.-L.G. designed experiments, analysed data and wrote the manuscript.
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K.-L.G. is a co-founder of and has an equity interest in Vivace Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.
Integrated supplementary information
Supplementary Figure 1 Osmotic stress induces TEAD cytoplasmic translocation.
(a) Time course of osmotic stress-induced TEAD cytoplasmic translocation. HEK293A cells were treated with NaCl for 0, 1, 3, or 6 h and stained for immunofluorescence. (b) Dose response for NaCl-induced TEAD cytoplasmic translocation. HEK293A cells were stimulated with different concentrations of NaCl for 6 h and stained for immunofluorescence. TEAD cytoplasmic translocation occurs from 100 mM NaCl. (c) High cell density-induced TEAD cytoplasmic localization is p38 independent. Immunofluorescence shows inhibition or KO of p38 upon high cell density has no effect on TEAD cytoplasmic translocation. (d) Cells ectopically expressing MKK3/p38 were stained for immunofluorescence. MKK3/p38 promotes TEAD4 cytoplasmic translocation. (e) Dose response for p38 inhibitor treatment. HEK293A cells were pre-treated with different doses of p38 inhibitors as indicated, followed by NaCl stimulation and stained for immunofluorescence. (f) Western blot of p38α/β knockout cells show upregulation of p38δ/γ isoforms. (g) p38 2KO cells were stained for immunofluorescence. Deletion of p38α/β isoforms is not sufficient to inhibit TEAD cytoplasmic translocation. (h) Immunoblotting for p-p38 in p38 4KO cells shows impaired p38 activity under various p38 activating stimuli (200 mM NaCl, 500 μM sorbitol, 500 μM arsenite). (i,j) p38 4KO shows no effect on TEAD localization (i) or target gene expression, as measured by qRT-PCR (j) under basal conditions. Scale bars in a–e, g, and i are 20 μm. Data are presented as mean ± s.e.m. from n = 3 independent experiments. Statistics source data are shown in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 5.
Supplementary Figure 2 Phosphorylation of TEAD by p38 is not required for cytoplasmic translocation.
(a) Sequence of TEAD4-4SP construct harboring mutations in putative p38 phosphorylation sites. (b–d) TEAD is a poor substrate for p38 phosphorylation. In vitro kinase assay for p38α (b), p38γ (c), and p38δ (d) using TEAD as a substrate. No phosphorylation or weak phosphorylation was detected in TEAD4 WT, which was further ablated in TEAD4-4SP, whereas ATF2 was effectively phosphorylated by p38. (e) Detection of TEAD4-4SP cytoplasmic translocation by p38 using immunofluorescence. (f) Sequence of putative TEAD nuclear export signal. (g) TEAD cytoplasmic translocation requires a nuclear export signal. Immunofluorescence shows truncation of TEAD disrupting a putative nuclear export signal in TEAD (1-382) mutant inhibits CRM1-dependent TEAD nuclear export compared to full length TEAD. (h) TEAD cytoplasmic translocation is a CRM1-dependent process. HEK293A cells were pretreated with LMB for the indicated times followed by NaCl stimulation and staining for immunofluorescence. Pretreatment with LMB inhibits TEAD cytoplasmic translocation. (i) Immunofluorescence of NFAT5 nuclear translocation upon NaCl stimulation. Scale bars in e and g–i are 20 μm. Unprocessed scans of blots are shown in Supplementary Fig. 5.
Supplementary Figure 3 Stress-induced TEAD cytoplasmic translocation is independent of Hippo pathway.
(a,b) Stress-induced TEAD cytoplasmic translocation is independent of Lats1/2. WT and Lats KO cells were stimulated with NaCl and stained with anti-TEAD1 (a) or anti-TEAD4 (b) antibody for immunofluorescence. (c,d,e) Stress-induced TEAD cytoplasmic translocation is independent of Hippo core kinases and MAP4K. Lats KO (c), Mst KO (d), and MAP4K KO (e) cells were treated with NaCl and stained for immunofluorescence. (f) Western blot showing MAP4K and p38 are independent branches of the MAPK pathway. (g) Western blot showing stress-activated p38 does not affect YAP phosphorylation status. Scale bars in a–e are 20 μm. Unprocessed scans of blots are shown in Supplementary Fig. 5.
Supplementary Figure 4 Stress-induced TEAD inhibition uncouples YAP localization and dephosphorylation in YAP-driven cancer cells.
(a,b) Stress-induced TEAD and YAP/TAZ cytoplasmic translocation in H2373 mesothelioma cells, which have homozygous deletion of NF2. Immunofluorescence showing NaCl stimulation induces TEAD and subsequent YAP/TAZ cytoplasmic sequestration (a), despite constitutive dephosphorylation of YAP as shown by western blot (b). NC, normal condition. (c) Nuclear localization of TEAD-VP16 in the presence of osmotic stress. MSTO-211H cells stably expressing TEAD1/4-VP16 construct were treated with NaCl and stained for immunofluorescence. (d,e) Stress promotes TEAD and YAP cytoplasmic sequestration in YAP-driven uveal melanoma cells. 92.1 cells were treated with YAP-inhibiting stimuli as in Fig. 1a, b. NaCl treatment elicits TEAD and YAP cytoplasmic translocation shown by immunofluorescence (d), despite constitutive dephosphorylation of YAP shown by western blot (e). (f,g) p38 mediates stress-induced TEAD cytoplasmic translocation in UM cell lines. Immunofluorescence shows treatment with SB203580 blocks NaCl-induced cytoplasmic translocation of TEAD in 92.1 (f) and OCM1 (g). (h) p38 expression inhibits colony formation of GNAQ-mutant 92.1 cells but not BRAF-mutant OCM1 cells. (i) Colony growth assay showing osmotic stress inhibits anchorage independent growth of YAP-5SA transformed MCF10A. (j) Expression of p38 reduces target gene expression induced by hyperactive YAP as measured by qRT-PCR. Target gene expression is rescued by constitutively active TEAD. Data are presented as mean from n = 2 independent experiments. (k–m) Immunohistochemistry staining of TEAD. Negative control staining for pan-TEAD antibody (left) and normal kidney tissue staining with pan-TEAD (right) (k). Nuclear staining of TEAD detected in mouse spleen and lung tissues (l). Cytoplasmic staining of TEAD is detected in tubule cells of normal kidney while nuclear staining is detected in renal clear cell carcinomas derived from transformed tubule cells (m). Scale bars in a, c,d, and f,g are 20 μm. Scale bars in k–m are 50 μm. Statistics source data are shown in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 5.
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Lin, K., Moroishi, T., Meng, Z. et al. Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation. Nat Cell Biol 19, 996–1002 (2017). https://doi.org/10.1038/ncb3581
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DOI: https://doi.org/10.1038/ncb3581
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