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Integrated RAS signaling defined by parallel NMR detection of effectors and regulators

Nature Chemical Biology volume 10, pages 223–230 (2014)Cite this article

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Abstract

The RAS GTPase directs cell proliferation and survival by selectively relaying signals amid a dynamic network of regulatory enzymes and protein interactions. Oncogenic mutation of RAS alters cell growth by deleteriously controlling output to RAS-binding effectors. Mechanisms underlying multieffector interactions for both wild-type and oncogenic RAS are poorly understood owing to challenges in quantifying outputs to multiple pathways in parallel. Using highly selective NMR probes for wild-type and oncogenic (G12V) RAS, we develop a systematic approach that quantitatively measures RAS output in composite mixtures of GEF, GAP and effector RAS-binding domains (RBDs). We derive effector signaling hierarchies and establish how oscillating concentrations generate effector 'switching'. The G12V mutation highly perturbs this system, specifically altering interactions with RAL GTPase-specific GEFs and RAF kinases. We further reveal that RAS–RBD complexes show extensive feedback to full-length regulatory proteins. Our approach quantifies output from signaling hubs, here providing an integrated view of the RAS network.

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Figure 1: Quantification of effector signaling by parallel detection of multiple RBD domains binding to [15N]RAS-GTP.
Figure 2: Establishing an RBD binding hierarchy.
Figure 3: RASG12V demonstrates distinct effector usage.
Figure 4: Wild-type RAS and RASG12V show opposing activation of RALA and ERK.
Figure 5: RBD–RAS complexes inhibit GAP activity.
Figure 6: RBDs inhibit full-length SOS1 by preventing its allosteric activation.

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Acknowledgements

We thank B. Neel, G. Findlay, C. Marshall and M. Mazhab-Jafari for valuable discussion and thoughts on the manuscript. This work was supported by a grant from the Cancer Research Society (to M.I.) and in part by the Canadian Cancer Society (to M.I.) and by the Princess Margaret Hospital Foundation. M.J.S. is the recipient of a Canadian Breast Cancer Foundation Fellowship and an Ontario Cancer Institute Knudson Postdoctoral Fellowship. M.I. holds a Canada Research Chair. The Canada Foundation for Innovation funded the 800- and 600-MHz NMR spectrometers.

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Authors and Affiliations

  1. Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada

    Matthew J Smith & Mitsuhiko Ikura

  2. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Mitsuhiko Ikura

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This project was conceived and designed by M.J.S. and M.I. All of the experimental work was performed by M.J.S. The manuscript was prepared by M.J.S. and M.I.

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Correspondence to Mitsuhiko Ikura.

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Smith, M., Ikura, M. Integrated RAS signaling defined by parallel NMR detection of effectors and regulators. Nat Chem Biol 10, 223–230 (2014). https://doi.org/10.1038/nchembio.1435

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