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STRIPAK integrates upstream signals to initiate the Hippo kinase cascade

Nature Cell Biology volume 21, pages 1565–1577 (2019)Cite this article

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A Publisher Correction to this article was published on 02 January 2020

This article has been updated

Abstract

The Hippo pathway plays a critical role in development, tissue homeostasis and organ size; its dysregulation contributes to human diseases. Although MST1/2 and the MAP4Ks are well known as the Hippo kinases, a major open question is how these kinases are regulated by upstream signals. Here we report that STRIPAK integrates upstream signals to control the activities of MST1/2 and the MAP4Ks, thus initiating Hippo signalling. STRIPAK also serves as a master regulator for the STE20 family kinases. Following serum or lysophosphatidic acid stimulation, active RhoA binds and dissociates rhophilin and NF2/Kibra from STRIPAK, thereby inducing the association and dephosphorylation of MST1/2 and MAP4Ks by the STRIPAK phosphatase catalytic subunit PP2AC. Rhophilin suppresses cancer cell growth by activating the Hippo pathway. Our study reveals a RhoA–rhophilin–NF2/Kibra–STRIPAK signalling axis in Hippo regulation, thus addressing the key question of how Hippo signalling is initiated and suggesting a broad and active role for STRIPAK in cellular signalling.

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Fig. 1: Serum and LPA regulate the interaction of MST1 or MAP4K4 with STRIPAK.
Fig. 2: STRIPAK complex is required for Hippo pathway regulation.
Fig. 3: Serum, LPA and RhoA regulate the kinase activity of MST1 and MAP4K4.
Fig. 4: RhoA mediates serum/LPA signal to regulate the MAP4K4–STRIPAK interaction and Hippo pathway.
Fig. 5: Kibra co-operates with NF2 to regulate the MAP4K4–STRIPAK interaction and Hippo signalling.
Fig. 6: RHPN acts downstream of RhoA to regulate MAP4K4–PP2AC interaction and Hippo signalling by controlling Kibra–NF2 interaction.
Fig. 7: RHPN1 inhibits colon cancer cell growth by activating the Hippo pathway.
Fig. 8: STRIPAK regulates the STE kinase family.

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Data availability

Source data for Figs. 1–8 and Extended Data Figs. 1–8 are available online. All other data that support the findings of this study are available on request from the corresponding author.

Change history

  • 02 January 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank S. Field for providing the STRN3 antibody. K.-L.G. is supported by grants from the National Institutes of Health (grant nos. CA196878, CA217642, GM51586, DEO15964).

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Authors and Affiliations

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

    Rui Chen, Ruiling Xie, Zhipeng Meng, Shenghong Ma & Kun-Liang Guan

  2. Department of Otolaryngology, Head & Neck Surgery, Peking University First Hospital, Beijing, China

    Ruiling Xie

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Contributions

R.C. and K.-L.G. designed the study, analysed the data and wrote the manuscript. R.C. and R.X. performed the experiments and collected data. Z.M. provided technical and intellectual support. S.M. provided intellectual support. All authors discussed the results and commented on the manuscript.

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Correspondence to Kun-Liang Guan.

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K.-L.G. is a cofounder of and has an equity interest in Vivace Therapeutics, Inc. 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.

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Extended data

Extended Data Fig. 1 Interaction between MST1, MST2, or MAP4K4 and STRIPAK components.

a, The phylogenetic tree of kinases tested in the interaction study. b-l, 293A cells were transiently transfected with the indicated plasmids of kinases and HA-STRIP1, HA-STRIP2, Myc-STRN1, Myc-STRN3, Myc-STRN4, Myc-SLMAP, Myc-CTTNBP2-NL, HA-CCM2, HA-CCM3, Myc-SIKE, or Myc-Mob4. Immunoprecipitated samples with anti-Flag antibody were subjected to immunoblot with the indicated antibodies. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for b-l are shown in Source Data Extended Data Figure 1.

Source Data

Extended Data Fig. 2 Interaction between MST1, MST2, or MAP4K4 and STRIPAK components continued.

a,b, 293A cells were transiently transfected with Flag-MST2 and HA-STRIP1, HA-STRIP2, Myc-STRN1, Myc-STRN3, Myc-STRN4, Myc-SLMAP, or Myc-CCM2. Immunoprecipitated samples with anti-Flag antibody were subjected to immunoblot. c,d, WT and STRIP1/2 KO cells were transiently transfected with Flag-MAP4K4 and Myc-STRN4 or Flag-STRN4 and HA-PP2AC for 24h, and then immunoprecipitated with anti-Flag antibody, and analyzed by immunoblot. e,f, WT and STRN1/3/4 KO cells were transfected with Flag-MAP4K4 and HA-STRIP1 or Flag-STRIP1 and HA-PP2AC for 24h, and then immunoprecipitated with anti-Flag antibody, analyzed by immunoblot. g,h, Quantification of PLA signals between 293A cells stably expressing Flag-MAP4K4 and HA-STRIP1 or stably expressing Flag-MAP4K4 and Myc-STRN4. i, 293A cells were transiently transfected with the Flag-STRN1 with Myc-SLMAP, HA-STRIP1 and HA-MST1. After 24h transfection, cells were serum-starved for 4h and then treat with serum for 0.5h. Flag-STRN1 immunoprecipitated from cell lysates was subjected to immunoblot. j, Summary of the interaction between kinases and the components of STRIPAK complex based on the co-immunoprecipitation experiments in Figure S1. “+” indicated positive interaction; “-” indicated negative interaction; “N/A” indicated not analyzed. k, The modified STRIPAK complex model. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a-f, i are shown in the Source Data Extended Data Fig. 2. Data in g, h are represented as the mean ± s.d. n=3 independent experiments. ~200 cells were monitored in each experiment. Statistical analysis was performed by one-way ANOVA (PRISM). **p < 0.01. Statistical source data for g, h is provided in Statistical Source Data Extended Data Fig. 2. Uncropped blots are shown in Source Data Extended Data Fig. 2.

Source Data

Extended Data Fig. 3 Flag-MST1 and Flag-MAP4K4 stable expressing cell lines; LPA does not regulate the interaction between MAP4K4 and STRIP1/2 or Striatins.

a,b, 293A cell were infected with lentivirus containing Flag-MST1 or Flag-MAP4K4. 24h after infection, cells were selected with 1.6 μg/ml puromycin to establish cell lines stably pools expressing Flag-MST1 or Flag-MAP4K4. The relative expressions of ectopic Flag-MST1 and Flag-MAP4K4 to endogenous proteins were detected by antibodies for MST1 and MAP4K4, respectively. c, 293A cells stably expressing Flag-MAP4K4 and HA-STRIP1 were serum-starved for 4h and then treated with 1μM LPA for different time points. Immunoprecipitated Flag-MAP4K4 was subjected to immunoblot. d, 293A cells stably expressing Flag-MAP4K4 and Myc-STRN4 were serum-starved for 4h and then treated with 1μM LPA. Immunoprecipitated Flag-MAP4K4 was subjected to immunoblot. e, 293A cells stably expressing Flag-MAP4K4 and Myc-SLMAP were serum-starved for 4h and then treated with 1μM LPA. Immunoprecipitated Flag-MAP4K4 was subjected to immunoblot. f, 293A cells stably expressing HA-STRIP1 were transiently transfected with Flag-STRN4 for 24h, and then serum-starved. Immunoprecipitated samples were subjected to immunoblot. g, 293A cells stably expressing Myc-STRN4 were serum-starved for 4h and then treated with 1μM LPA. Immunoprecipitated Myc-STRN4 was subjected to immunoblot. h, 293A cells stably expressing HA-STRIP1 were serum-starved. Immunoprecipitated samples were subjected to immunoblot. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a-h are shown in Source Data Extended Data Figure 3.

Source Data

Extended Data Fig. 4 Okadaic acid increases phosphorylation of MST1, LATS1, and YAP; and establishment of in vitro kinase assay.

a, 293A cells were treated with different concentrations of Okadaic Acid (OA) for 4h and then subjected to phos-tag and immunoblot analysis. b, SLMAP deletion partially compromises Hippo regulation by LPA. WT and two independent SLMAP KO clones were serum-starved for 4h and then treated with 1μM LPA. The effects on Hippo signaling were determined. c, Sequencing of STRIP2 knockout cell lines. d, STRIP1/2 knockout abolishes Hippo regulation by LPA. WT and STRIP1/2 KO clone #7 were serum-starved for 4h and then treated with 1μM LPA. The effects on Hippo signaling were examined by Western blot. e, Sequencing of STRN1 knockout cell lines. f, The scheme of the in vitro kinase assays for MST1 MAP4K4. g, 293A cells stably expressing Flag-MST1 were serum-starved or stimulated as indicated. Immunoprecipitated Flag-MST1 was subjected to in vitro kinase assay using full-length GST-LATS2 or truncated GST-LATS1 as a substrate. Phosphorylation of LATS was determined by immunoblotting using pLATS-HM antibody. h, WT and STRIP1/2 KO cells stably expressing Flag-MST1 were immunoprecipitated with Flag antibody, one to three dilution of Flag-MST1 kinase was subjected to in vitro kinase assay. The star (*) near the 55kDa indicates a non-specific band. i, The phosphorylation of MST1 in wt and STRIP1/2 KO with one to three dilution of cell lysate. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a, b, d, g-i are shown in Source Data Extended Data Figure 4.

Source Data

Extended Data Fig. 5 LPA does not affect dimer formation of MAP4K4 or MST1.

a, 293A cells were serum-starved for 4h and treated with 1% LPA, and then subjected to immunoblot. b,c, 293A cells stably expressing Flag-MST1 or Flag-MAP4K4 were transiently transfected with HA-MST1 or HA-MAP4K4. Cells were serum-starved for 4h and then treated with 1μM LPA. Immunoprecipitated Flag-MST1 or Flag-MAP4K4 was subjected to immunoblot with the indicated antibodies. d, 293A cells were serum-starved for 4h and treated with 1% serum. Endogenous MST1 was detected by pMST1 antibody. e, 293A cells stably expressing GST-MST1 were serum-starved for 4h and then treated with 1%serum for 0.5h. Immunoprecipitated GST-MST1 was subjected to WB with phosphorylated MST1 antibody. f, 293A cells transiently transfected with Flag-MAP4K4, Flag-MAP4K4 (K54R) or Flag-MAP4K4 (T191A) were serum-starved for 4h and then treated with 1μM LPA. Immunoprecipitated Flag-MAP4K4 was subjected to in vitro kinase assay. g, 293A cells were serum-starved for 1h and treated with serum or LPA in the presence of Y27632 (ROCK inhibitor) or not, and then subjected to immunoblot. h, 293A cells were transiently transfected with Myc-RhoA and Flag-YAP in the Y27632 or not under serum starvation, cell lysates were subjected to immunoblot. i, Quantification of PLA signals between Flag-MST1 and PP2AC in WT and STRIP1/2 KO cells stably expressing Flag-MST1 treated with or without LPA. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a-h are shown in Source Data Extended Data Fig. 5. Data in i are represented as the mean ± s.d. n=3 independent experiments. ~200 cells were monitored in each experiment. Statistical analysis was performed by one-way ANOVA (PRISM). **p < 0.05, **p < 0.01. Statistical source data for i is provided in Statistics Source Data Extended Data Fig. 5.

Source Data

Extended Data Fig. 6 The interaction of Hippo pathway components with SLMAP or STRN4; verification of WWC1/2/3 knockout cell lines.

a, 293A cells were transiently transfected with Myc-STRN4 and the indicated plasmids, immunoprecipitated samples with anti-Flag antibody were subjected to immunoblot for their interaction with STRN4. b, 293A cells were transiently transfected with Myc-SLMAP and the indicated plasmids, immunoprecipitated samples with anti-Flag antibody were subjected to immunoblot. c, 293A cells were transiently transfected with HA-Kibra and Flag-NF2, and then HA-Kibra immunoprecipitated from cell lysates was subjected to immunoblot. d, Genomic DNA sequencing of the Kibra (WWC1), WWC2 and WWC3 knockout cell line. e, Wild type or MM8KO (combined deletion of two MST genes and six MAP4K genes) cells were transfected with Flag-YAP in the presence or absence of HA-Kibra and HA-NF2. YAP phosphorylation was determined by phostag gel. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a-c, e are shown in Source Data Extended Data Figure 6.

Source Data

Extended Data Fig. 7 RHPN1 regulates MAP4K4-PP2AC interaction and Hippo pathway; verification of RHPN1/2 knockout cell line.

a, 293A cells were transiently transfected with Myc-RhoA, HA-RHPN1, or HA-RHPN2. Immunoprecipitated Myc-RhoA was subjected to immunoblot. b, WT or RhoA KO cells with stably expression of Flag-MAP4K4 were transiently transfected with HA-RHPN1. Immunoprecipitated Flag-MAP4K4 was subjected to immunoblot. c, Genomic DNA sequencing of the RHPN1 and RHPN2 knockout cell line. d, 293A cells were transiently transfected with Flag-Kibra and wild type or mutant construct of RhoA in the presence of HA-RHPN1. Immunoprecipitated Flag-Kibra was subjected to immunoblot. e, 293A cells stably expressing Flag-Kibra were serum-starved for 4 h and then treated with 1μM LPA. Immunoprecipitated Flag-Kibra was subjected to immunoblot. f, Quantification of PLA signals between Flag-Kibra and PP2AC in 293A cells stably expressing Flag-Kibra treated with or without LPA. g, Quantification of PLA signals between Flag-Kibra and HA-STRIP1 in 293A cells stably expressing Flag-Kibra and HA-STRIP1 treated with or without LPA. h, 293A cells were transiently transfected with HA-RHPN1 and Myc-RhoA in the presence of Flag-YAP under serum starvation, cell lysates were subjected to immunoblot. i, WT or RHPN1/2 KO cells were transiently transfected with Flag-YAP with or without Myc-RhoA N19. Cell lysates were analyzed by immunoblot. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for a, b, d, e, h, i are shown in Source Data Extended Data Fig. 7. Data in f, g are represented as the mean ± s.d. n=3 independent experiments. ~200 cells were monitored in each experiment. Statistical analysis was performed by one-way ANOVA (PRISM). **p < 0.01. Statistical source data for f, g is provided Statistics Source Data Extended Data Fig. 7.

Source Data

Extended Data Fig. 8 The expression of RHPN1 in colon cancer cell line and STRIP1/2 deletion enhances phosphorylation of MST3/MST4/YSK1.

a, The mRNA expression of RHPN1 in colon cancer cell lines (Cancer Cell Line Encyclopedia (CCLE) database). b, HCT 116 and HCT116 stably expressing Myc-RHPN1 were transiently transfected with Flag-STRIP1 as indicated, after 48h transfection, cells were serum-starved for 4h and then treat with serum for 0.5h. Flag-STRIP1 immunoprecipitated from cell lysates was subjected to immunoblot. c, WT and STRIP1/2 KO cells were serum-starved for 4h and then treated with 1μM LPA for different time points. Cell lysates were subjected to immunoblot with phosphorylated MST3/MST4/YSK1 antibody and other indicated antibodies. All experiments were repeated independently at least three times with similar results. Uncropped images of blots for b, c are shown in the Source Data Extended Data Figure 8.

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Supplementary Information

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Chen, R., Xie, R., Meng, Z. et al. STRIPAK integrates upstream signals to initiate the Hippo kinase cascade. Nat Cell Biol 21, 1565–1577 (2019). https://doi.org/10.1038/s41556-019-0426-y

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