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Divergent effects of intrinsically active MEK variants on developmental Ras signaling

Nature Genetics volume 49, pages 465–469 (2017)Cite this article

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Abstract

Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays1,2. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown3,4. We used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans5,6. Surprisingly, we discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling.

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Figure 1: MEK variants are constitutively active in vitro.
Figure 2: MEK variants cause divergent effects on ERK activation in vivo.
Figure 3: Opposing effects of activating MEK mutations on ERK-dependent morphological phenotypes.
Figure 4: A two-input mathematical model for feedback-induced effects on ERK signaling.
Figure 5: A feedback-based mathematical model.
Figure 6: MEK variants cause divergent effects on ERK signaling in zebrafish.

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Acknowledgements

We thank C. Hasty, P. Johnson, J. London, and LAR staff for zebrafish care; A. Veraksa (University of Massachusetts, Boston), R. Seger (Weizmann Institute), and E. Goldsmith (UT Southwestern) for wild-type MEK and ERK constructs; M. Cardoso for help with the FISH probes; and G. Laevsky and the Molecular Biology Confocal Microscopy Facility, which is a Nikon Center of Excellence, for microscopy support. We thank E. Goldsmith, A. Veraksa, B. Gelb, R. Seger, J. Humphreys, and H. Mattingly for helpful discussions. S.Y.S. and A.S.F. thank J. Link for his input during the initial stages of this project. S.Y.S. and Y.G. thank V. Zini for her input during the initial stages of this project. Y.G., K.Y., E.Y., A.S.F., and S.Y.S. were supported by National Institutes of Health grant R01 GM086537. G.A.J. acknowledges support from an NSF Graduate Research Fellowship under grant DGE 1148900. R.D.B. was supported by National Institutes of Health grants R01 HD048584 and R01GM086537. J.L.P. was supported by National Institutes of Health grant R01 HD048584. T.S. was supported by National Institutes of Health grant R01 GM077620.

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Author notes

  1. Yogesh Goyal and Granton A Jindal: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA

    Yogesh Goyal, Granton A Jindal, Alan S Futran & Stanislav Y Shvartsman

  2. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA

    Yogesh Goyal, Granton A Jindal, Kei Yamaya, Eyan Yeung, Alan S Futran & Stanislav Y Shvartsman

  3. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA

    Yogesh Goyal, Granton A Jindal, José L Pelliccia, Kei Yamaya, Eyan Yeung, Rebecca D Burdine, Trudi Schüpbach & Stanislav Y Shvartsman

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Contributions

Y.G., G.A.J., R.D.B., T.S., and S.Y.S. conceived and designed the project. Y.G., E.Y., A.S.F., and G.A.J. performed in vitro experiments; Y.G. and K.Y. performed experiments in Drosophila; Y.G. and S.Y.S. developed the model with inputs from G.A.J.; and G.A.J. and J.L.P. performed experiments in zebrafish. Y.G. and G.A.J. analyzed the results. Y.G., G.A.J., and S.Y.S. wrote the manuscript with input from R.D.B. and T.S.

Corresponding author

Correspondence to Stanislav Y Shvartsman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Table 3 (PDF 3076 kb)

Supplementary Table 1

Quantification of dpERK/MEK and dpERK/ERK for four experimental replicates of the in vitro phosphorylation reactions for a 1:5 MEK:ERK ratio. (XLSX 16 kb)

Supplementary Table 2

Quantification of dpERK/MEK and dpERK/ERK in the in vitro phosphorylation reaction for a 1:10 ratio of MEK:ERK. (XLSX 11 kb)

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Goyal, Y., Jindal, G., Pelliccia, J. et al. Divergent effects of intrinsically active MEK variants on developmental Ras signaling. Nat Genet 49, 465–469 (2017). https://doi.org/10.1038/ng.3780

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