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Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling

Nature Cell Biology volume 16, pages 673–684 (2014)Cite this article

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Abstract

Signal transduction requires the coordination of activities between different pathways. In mammalian cells, Raf-1 regulates the MST–LATS and MEK–ERK pathways. We found that a complex circuitry of competing protein interactions coordinates the crosstalk between the ERK and MST pathways. Combining mathematical modelling and experimental validation we show that competing protein interactions can cause steep signalling switches through phosphorylation-induced changes in binding affinities. These include Akt phosphorylation of MST2 and a feedback phosphorylation of Raf-1 Ser 259 by LATS1, which enables Raf-1 to suppress both MST2 and MEK signalling. Mutation of Raf-1 Ser 259 stimulates both pathways, simultaneously driving apoptosis and proliferation, whereas concomitant MST2 downregulation switches signalling to cell proliferation, transformation and survival. Thus, competing protein interactions provide a versatile regulatory mechanism for signal distribution through the dynamic integration of graded signals into switch-like responses.

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Figure 1: Crosstalk between MST2 and ERK pathways.
Figure 2: Competing protein interactions can cause switch-like transitions.
Figure 3: LATS1 phosphorylation of Raf-1 Ser 259 regulates MST2–Raf-1 complex formation.
Figure 4: A mathematical model for MST2 and ERK pathway crosstalk.
Figure 5: Model analysis and experimental validation.
Figure 6: The LATS1 feedback loop regulates ERK pathway activation.
Figure 7: RASSF1A regulation of MST2 and ERK activation.
Figure 8: MST2 and ERK pathway crosstalk regulates cell proliferation, survival and transformation.

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References

  1. Kholodenko, B. N., Hancock, J. F. & Kolch, W. Signalling ballet in space and time. Nat. Rev. Mol. Cell Biol. 11, 414–426 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jorgensen, C. & Linding, R. Simplistic pathways or complex networks? Curr. Opin. Genet. Dev. 20, 15–22 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179–185 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C. & Blenis, J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat. Cell Biol. 4, 556–564 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Nakakuki, T. et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141, 884–896 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Von Kriegsheim, A. et al. Cell fate decisions are specified by the dynamic ERK interactome. Nat. Cell Biol. 11, 1458–1464 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Matallanas, D. et al. Raf family kinases: old dogs have learned new tricks. Genes Cancer 2, 232–260 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Osborne, J. K., Zaganjor, E. & Cobb, M. H. Signal control through Raf: in sickness and in health. Cell Res. 22, 14–22 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Chen, J., Fujii, K., Zhang, L., Roberts, T. & Fu, H. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism. Proc. Natl Acad. Sci. USA 98, 7783–7788 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. O’Neill, E., Rushworth, L., Baccarini, M. & Kolch, W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267–2270 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Ehrenreiter, K. et al. Raf-1 regulates Rho signaling and cell migration. J. Cell Biol. 168, 955–964 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Avruch, J. et al. Protein kinases of the Hippo pathway: regulation and substrates. Semin. Cell Dev. Biol. 23, 770–784 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Visser, S. & Yang, X. LATS tumor suppressor: a new governor of cellular homeostasis. Cell Cycle 9, 3892–3903 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Matallanas, D. et al. RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol. Cell 27, 962–975 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Richter, A. M., Pfeifer, G. P. & Dammann, R. H. The RASSF proteins in cancer; from epigenetic silencing to functional characterization. Biochim. Biophys. Acta 1796, 114–128 (2009).

    CAS  PubMed  Google Scholar 

  16. Avruch, J. et al. Rassf family of tumor suppressor polypeptides. J. Biol. Chem. 284, 11001–11005 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Romano, D. et al. Proapoptotic kinase MST2 coordinates signaling crosstalk between RASSF1A, Raf-1, and Akt. Cancer Res. 70, 1195–1203 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Halder, G. & Johnson, R. L. Hippo signaling: growth control and beyond. Development 138, 9–22 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bossuyt, W. et al. An evolutionary shift in the regulation of the Hippo pathway between mice and flies. Oncogene 33, 1218–1228 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. White, M. A. et al. Multiple Ras functions can contribute to mammalian cell transformation. Cell 80, 533–541 (1995).

    Article  CAS  PubMed  Google Scholar 

  21. Rodriguez-Viciana, P. et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457–467 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Kubicek, M. et al. Dephosphorylation of Ser-259 regulates Raf-1 membrane association. J. Biol. Chem. 277, 7913–7919 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Dhillon, A. S., Meikle, S., Yazici, Z., Eulitz, M. & Kolch, W. Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J. 21, 64–71 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jaumot, M. & Hancock, J. F. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions. Oncogene 20, 3949–3958 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Dhillon, A. S. et al. Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259. Mol. Cell. Biol. 22, 3237–3246 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Dumaz, N. & Marais, R. Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras. J. Biol. Chem. 278, 29819–29823 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Dhillon, A. S., von Kriegsheim, A., Grindlay, J. & Kolch, W. Phosphatase and feedback regulation of Raf-1 signaling. Cell Cycle 6, 3–7 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Markevich, N. I., Hoek, J. B. & Kholodenko, B. N. Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J. Cell Biol. 164, 353–359 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sturm, O. E. et al. The mammalian MAPK/ERK pathway exhibits properties of a negative feedback amplifier. Sci. Signal. 3, ra90 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Dougherty, M. K. et al. Regulation of Raf-1 by direct feedback phosphorylation. Mol. Cell 17, 215–224 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Anand, R., Kim, A. Y., Brent, M. & Marmorstein, R. Biochemical analysis of MST1 kinase: elucidation of a C-terminal regulatory region. Biochemistry 47, 6719–6726 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Glantschnig, H., Rodan, G. A. & Reszka, A. A. Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation. J. Biol. Chem. 277, 42987–42996 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Hamilton, G., Yee, K. S., Scrace, S. & O’Neill, E. ATM regulates a RASSF1A-dependent DNA damage response. Curr. Biol. 19, 2020–2025 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Praskova, M., Khoklatchev, A., Ortiz-Vega, S. & Avruch, J. Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras. Biochem. J. 381, 453–462 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Guo, C., Zhang, X. & Pfeifer, G. P. The tumor suppressor RASSF1A prevents dephosphorylation of the mammalian STE20-like kinases MST1 and MST2. J. Biol. Chem. 286, 6253–6261 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chan, E. H. et al. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24, 2076–2086 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Ferrell, J. E. Jr & Machleder, E. M. The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. Science 280, 895–898 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Dhillon, A. S. et al. A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation. Mol. Cell. Biol. 23, 1983–1993 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Miesfeld, J. B. & Link, B. A. Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway. Mech. Dev. (2014)10.1016/j.mod.2014.02.003

  40. Sha, W. et al. Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts. Proc. Natl Acad. Sci. USA 100, 975–980 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Qiao, L., Nachbar, R. B., Kevrekidis, I. G. & Shvartsman, S. Y. Bistability and oscillations in the Huang–Ferrell model of MAPK signaling. PLoS Comput. Biol. 3, 1819–1826 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Kim, S. Y. & Ferrell, J. E. Jr Substrate competition as a source of ultrasensitivity in the inactivation of Wee1. Cell 128, 1133–1145 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Kholodenko, B. N. Cell-signalling dynamics in time and space. Nat. Rev. Mol. Cell Biol. 7, 165–176 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Salazar, C. & Hofer, T. Competition effects shape the response sensitivity and kinetics of phosphorylation cycles in cell signaling. Ann. NY Acad. Sci. 1091, 517–530 (2006).

    Article  PubMed  Google Scholar 

  45. Askew, D. S., Ashmun, R. A., Simmons, B. C. & Cleveland, J. L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 6, 1915–1922 (1991).

    CAS  PubMed  Google Scholar 

  46. Evan, G. I. et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119–128 (1992).

    Article  CAS  PubMed  Google Scholar 

  47. Matallanas, D. et al. Mutant K-Ras activation of the proapoptotic MST2 pathway is antagonized by wild-type K-Ras. Mol. Cell 44, 893–906 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Pandit, B. et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat. Genet. 39, 1007–1012 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Razzaque, M. A. et al. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat. Genet. 39, 1013–1017 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Kobayashi, T. et al. Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation. Hum. Mutat. 31, 284–294 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Geiger, T., Wehner, A., Schaab, C., Cox, J. & Mann, M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins. Mol. Cell. Proteom. 11, M111 014050 (2012).

    Article  CAS  Google Scholar 

  52. Dent, P., Reardon, D. B., Morrison, D. K. & Sturgill, T. W. Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. Mol. Cell. Biol. 15, 4125–4135 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ortiz-Vega, S. et al. The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1. Oncogene 21, 1381–1390 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Luzyanina, T. et al. Numerical modelling of label-structured cell population growth using CFSE distribution data. Theor. Biol. Med. Model. 4, 26 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio) 4th edn (Univ. Oregon Press, 2000).

    Google Scholar 

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Acknowledgements

This work was supported by Science Foundation Ireland grant No. 06/CE/B1129 and the EU FP7 Projects PRIMES No. 278568 and ASSET No. 259348-2. We thank A. Blanco (Conway Core Technologies), T. Santra, J. Munoz and M. Dobrzynski for help with flow cytometry and modelling.

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Author notes

  1. Boris N. Kholodenko and Walter Kolch: These authors jointly supervised this work.

Authors and Affiliations

  1. Systems Biology Ireland, University College Dublin, Dublin 4, Ireland

    David Romano, Lan K. Nguyen, David Matallanas, Melinda Halasz, Carolanne Doherty, Boris N. Kholodenko & Walter Kolch

  2. Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin 4, Ireland

    Boris N. Kholodenko & Walter Kolch

  3. School of Medicine and Medical Sciences, University College Dublin, Dublin 4, Ireland

    Boris N. Kholodenko & Walter Kolch

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Contributions

D.R., D.M., C.D. and M.H. performed the experiments, L.K.N. and B.N.K. performed the modelling, W.K. directed the overall research and all authors wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 The MST2—Raf-1 interaction.

(a) Disruption of the MST2—Raf-1 protein complex by peptides in vitro. 10 ng of purified GST-Raf-1 protein were incubated with 1 μg of recombinant MST2 in the presence or absence of N-terminal stearylated disruptor or scrambled control peptides. GST pulldowns were analyzed by Western blotting as indicated. The sequence of the disruptor peptide is given below with the 17 amino acid interaction domain indicated in bold. (b) Co-immunoprecipitation of known Raf-1 interactors is not disturbed by the disruptor peptide. Serum starved HeLa cells were incubated with stearoylated-disruptor or scrambled control peptides. Raf-1 immunoprecipitates (IPs) were Western blotted for the known Raf-1 binding proteins KSR1, HSP90, and 14-3-3. (c) Mapping of the MST2 interaction sites on Raf-1. The Raf-1 amino acid sequence was synthesized as a set of 23mer peptides overlapping by 5 amino acids that were immobilised on a nitrocellulose membrane. The membrane was probed with a 35S-methionine labelled MST2 protein that was produced by coupled in vitro transcription translation as described in the methods section. MST2 binding peptides in the Raf-1 sequence are boxed in red, and the MST2 binding sites are indicated on the schematic Raf-1 structure.

Supplementary Figure 2 Protein concentrations and effects on Raf-1 binding to MST2 or MEK.

(a,b) Determination of Raf-1, MEK and MST2 protein concentrations by quantitative Western blotting. (a) Coomassie stained gel of purified, recombinant proteins used as standards. (b) Examples of Western blots where the concentrations of Raf-1, MEK, and MST2 in MCF7 cells were determined by comparison of the signal obtained in cell lysates against respective purified protein standards of known concentrations. Blots were quantified by laser densitometry and analysed using the NIH Image J software. The standards were used to generate a reference curve, and the concentration of the proteins in the cell lysates were calculated after adjustment for the dilution during cell lysis. The table shows protein concentrations per cell assuming a cell volume of 1pL. (c) MST2 and MEK compete for Raf-1 binding in vitro. 1 ng of recombinant GST-tagged Raf-1 was incubated in vitro with 2 ng of recombinant MEK and increasing amounts (0.01–2 ng) of recombinant MST2. GST pulldowns and total levels of proteins were analyzed by Western blot using antibodies against the indicated proteins. Blots were quantified by laser densitometry as above.

Supplementary Figure 3 The role of Raf-1 S259 phosphorylation.

(a) Simultaneous activation of Raf-1 and MST2 signaling by S259 dephosphorylation and RASSF1A. Raf-1−/− MEFs were co-transfected with either wild type (wt) Flag-Raf-1 or the Flag-Raf-1 S259A mutant and HA-RASSF1A as indicated. Flag-IPs were analyzed by Western blotting using antibodies against the indicated proteins. MST2 kinase activity was assayed from MST2 IPs by an in gel kinase assay as described in the Methods section. 10 μg of cell extracts were blotted using antibodies against phospho- or total proteins as indicated. (b) Mutation of Ser 338/339 in Raf-1 does not affect binding to MST2. MCF7 cells were co-transfected with Flag-Raf-1 or the Flag-Raf-1 SS338/9 or S259A mutants as indicated. Flag-IPs were blotted for associated MST2 and MST2 IPs were blotted with Flag antibody. (c) Inhibition of phosphoinositide-3 kinase (PI3K) does not affect Raf-1 phosphorylation on S259. MCF7 cells were treated with 10 μM PI3K inhibitor LY294002 (LY) for the indicated times. MST2 IPs were examined for associated Raf-1 and phosphorylation of S259 in Raf-1. 10 μg cell extracts were blotted for Raf-1 and pS259. Blots were quantified as above. The graph shows the S259 phosphorylation in extracts normalized to Raf-1 levels in the extracts; and the Raf-1 and pS259 signals in MST2 IPs normalized to the levels of MST2, or Raf-1 and MST2 contained in the IPs, respectively. The dissociation of the MST2—Raf-1 complex is due to the inhibition of Akt mediated MST2 phosphorylation, which enhances the binding of MST2 to Raf-11. (d) LATS1 downregulation induces a switch-like change from MST2–Raf-1 to MEK-Raf-1 complexes and an activation of Raf-1, ERK and MST2. Hela cells were transfected with increasing concentration of LATS1 (0–100 nM) or scrambled control (C) siRNAs. Raf-1 and MST2 IPs and 10 μg of cellular extracts were analyzed by Western blotting for the indicated phospho- and total proteins. Blots were quantitated by laser densitometry and analysed using the Image J software. Corresponding simulations are shown in Fig. M8.

Supplementary Figure 4 Activated PI3 kinase synergises with the Raf-1 S259A in cell transformation.

NIH3T3 cells were transfected with the indicated constructs, allowed to grow to confluence and assayed for focus formation 2 weeks after transfection. p110a is the catalytic subunit of PI3Kα rendered constitutively active by fusion to a myristylation signal. BXB is the truncated Raf-1 kinase domain, which functions as oncogene2. Error bars represent s.e.m., n = 4.

Supplementary Figure 5 Schematic summary.

Switch-like transitions between Raf-1 binding to MST2 or MEK determine biological outcomes such as proliferation versus apoptosis.

Supplementary Figure 6 Uncropped images of films of key experiments.

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Romano, D., Nguyen, L., Matallanas, D. et al. Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling. Nat Cell Biol 16, 673–684 (2014). https://doi.org/10.1038/ncb2986

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