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

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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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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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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Correspondence to Boris N. Kholodenko or Walter Kolch.

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The authors declare no competing financial interests.

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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