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Nat Chem Biol. 2014 Mar;10(3):223-30. doi: 10.1038/nchembio.1435. Epub 2014 Jan 19.

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

Author information

1
Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada.
2
1] Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada. [2] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

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.

PMID:
24441586
DOI:
10.1038/nchembio.1435
[Indexed for MEDLINE]

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