Using Engineered Scaffold Interactions to Reshape MAP Kinase Pathway Signaling Dynamics

Department of Cellular and Molecular Pharmacology, University of California at San Francisco, 600 16th Street, San Francisco, CA 94158, USA.
Science (Impact Factor: 33.61). 04/2008; 319(5869):1539-43. DOI: 10.1126/science.1151153
Source: PubMed


Scaffold proteins link signaling molecules into linear pathways by physically assembling them into complexes. Scaffolds may
also have a higher-order role as signal-processing hubs, serving as the target of feedback loops that optimize signaling amplitude
and timing. We demonstrate that the Ste5 scaffold protein can be used as a platform to systematically reshape output of the
yeast mating MAP kinase pathway. We constructed synthetic positive- and negative-feedback loops by dynamically regulating
recruitment of pathway modulators to an artificial binding site on Ste5. These engineered circuits yielded diverse behaviors:
ultrasensitive dose response, accelerated or delayed response times, and tunable adaptation. Protein scaffolds provide a flexible
platform for reprogramming cellular responses and could be exploited to engineer cells with novel therapeutic and biotechnological

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    • "Engineering signalling pathways has significant potential to provide novel and complementary information that cannot be achieved by traditional genetic approaches such as gene knockout and overexpression1112. For instance, rewiring signalling components between MAPK pathways1314, introducing synthetic negative or positive feedback loops1516, tethering signalling components with specific localization motifs17, assembling or recombining modular signalling domains1819 and reconstitution of a heterologous MAPK cascade20 are highly informative for understanding the design principles of MAPK pathways and enable generating novel signalling properties. In the present study, we reconstituted osmoadaptation in hog1Δ cells by rewiring osmostress signalling through the MAPK network. "
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    ABSTRACT: Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data.
    Full-text · Article · Apr 2014 · Scientific Reports
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    • "To further optimize this trade-off at the combination design stage, we propose that other means of customizing the I/O response to improve drug resistance potential are needed, e.g., by using the methods and results of synthetic biology in signaling pathway engineering to customize their I/O responses (69–71). For example, using some modulators it is possible to alter/reshape dose dependence from a graded to a sharply sensitive, switch-like response and a time dependence from sustained to pulse or delayed responses in MAPK pathway signaling (71). It has been shown that modification of cellular response obtained by genetic engineering can also be reached through drug action. "
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    ABSTRACT: Drug resistance, de novo and acquired, pervades cellular signalling networks from one signalling motif to another as a result of cancer progression and/or drug intervention. This resistance is one of the key determinants of efficacy in targeted anticancer drug therapy. Although poorly understood, drug resistance is already being addressed in combination therapy by selecting drug targets where sensitivity increases due to combination components or as a result of de novo or acquired mutations. Additionally, successive drug combinations have shown low resistance potency. To promote a rational, systematic development of combination therapies, it is necessary to establish the underlying mechanisms that drive the advantages of drug combinations and design methods to determine advanced targets for drug combination therapy. Based on a joint systems analysis of cellular signalling network (SN) response and its sensitivity to drug action and oncogenic mutations, we describe an in silico method to analyse the targets of drug combinations. The method explores mechanisms of sensitizing the SN through combination of two drugs targeting vertical signalling pathways. We propose a paradigm of SN response customization by one drug to both maximize the effect of another drug in combination and promote a robust therapeutic response against oncogenic mutations. The method was applied to the customization of the response of the ErbB/PI3K/PTEN/AKT pathway by combination of drugs targeting HER2 receptors and proteins in the downstream pathway. The results of a computational experiment showed that the modification of the SN response from hyperbolic to smooth sigmoid response by manipulation of two drugs in combination leads to greater robustness in therapeutic response against oncogenic mutations determining cancer heterogeneity. The application of this method in drug combination co-development suggests a combined evaluation of inhibition effects along with the capability of drug combinat
    Full-text · Article · Feb 2014 · Frontiers in Oncology
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    • "In the cytosol, signals can transmit through the large assemblies, such as the nucleosomes in the nucleus; and as we argue here, also through the structured cytoskeleton, which is similarly dynamic. In all cases, scaffolding proteins [103] [105] [106] which are sometimes overlooked in cellular diagrams are likely to play major roles. Scaffolding proteins do not communicate the signal passively; they can control it [103]. "
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    ABSTRACT: The spatial structure of the cell is highly organized at all levels: from small complexes and assemblies, to local nano- and microclusters, to global, micrometer scales across and between cells. We suggest that this multiscale spatial cell organization also organizes signaling and coordinates cellular behavior. We propose a new view of the spatial structure of cell signaling systems. This new view describes cell signaling in terms of dynamic allosteric interactions within and among distinct, spatially organized transient clusters. The clusters vary over time and space and are on length scales from nanometers to micrometers. When considered across these length scales, primary factors in the spatial organization are cell membrane domains and the actin cytoskeleton, both also highly dynamic. A key challenge is to understand the interplay across these multiple scales, link it to the physicochemical basis of the conformational behavior of single molecules and ultimately relate it to cellular function. Overall, our premise is that at these scales, cell signaling should be thought of not primarily as a sequence of diffusion-controlled molecular collisions, but instead transient, allostery-driven cluster re-forming interactions.
    Preview · Article · Aug 2013 · Physical Biology
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