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

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.DOI: http://dx.doi.org/10.7554/eLife.12435.001

Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE’s membrane targeting sequence

“…Thus, it is likely that new dynamic behaviors will be uncovered and that similar effects to the ones here can be obtained through different kinds of perturbations. Interestingly, a trade-off between versatile dynamic behavior and sensitivity to biochemical changes has recently been reported for a reconstituted Ras signaling network [ 41 ], suggesting that this phenomenon might be widespread. The exploration of such trade-offs in biochemical networks as well as their careful balancing in the context of synthetic cell designs are intriguing future prospects.…”

Ciliate behavior: blueprints for dynamic cell biology and microscale robotics

“…This echoes how different biochemical activities and targeting domains have been harnessed to diversify cell signaling outputs ( Bhattacharyya et al. , 2006 ; Coyle and Lim, 2016 ), or how the Hox genes have been used to diversify body plans in animals ( Dassow and Munro, 1999 ).…”

Distinct oncogenic Ras signals characterized by profound differences in flux through the RasGDP/RasGTP cycle

“…In addition, they could also show that oncogenic Ras MUT still depends on the RasGEF/RasGAP circuit further shaping the Ras signaling outcome. 51 Development of small molecules that specifically bind to the Ras MUT KRAS G12C and decrease the viability of cancer cells with this mutation led to the realization that there is a pocket in KRAS G12C that may be a good target for cancer therapy. 52 Two subsequent studies reveal that KRAS G12C is still subjective to nucleotide exchange and does not fully get "locked in" it s constitutively active form.…”