Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

Author:

Coyle Scott M1234,Lim Wendell A1234

Affiliation:

1. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States

2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

3. Program in Biological Sciences, University of California, San Francisco, San Francisco, United States

4. Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, United States

Abstract

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

Funder

National Institutes of Health

Howard Hughes Medical Institute

National Science Foundation

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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