Integrated RAS signaling defined by parallel NMR detection of effectors and regulators.
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.
Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada.
Matthew J
MJ
Smith
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.
Mitsuhiko
M
Ikura
D000494:Allosteric Regulation; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry; D015398:Signal Transduction; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D020690:GTPase-Activating Proteins; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins