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. 2011 Jan 7;144(1):119-31.
doi: 10.1016/j.cell.2010.12.014.

Tunable signal processing in synthetic MAP kinase cascades

Ellen C O'Shaughnessy  1 , Santhosh Palani, James J Collins, Casim A Sarkar
Affiliations

Tunable signal processing in synthetic MAP kinase cascades

Ellen C O'Shaughnessy et al. Cell. .

Abstract

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems.

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Figures

Figure 1
Figure 1. The Basic Synthetic Cascade
A. Schematic of the basic synthetic cascade. Raf:ER, MEK, and ERK were co-expressed by PtetO7. The kinase activity of Raf:ER is modulated by estradiol. See also Figure S1A. B. Representative western blot of steady-state ERK activation with increasing estradiol concentration. C. ERK activation in response to estradiol titration (black squares). The experimental data are the mean ± SEM normalized to the fitted baselines. Data were fit with a modified Hill equation (black line). The model simulation results of the steady-state response profile for the basic cascade are shown as a red line. The system response is moderately ultrasensitive with a Hill coefficient of 1.8 ± 0.13 and an EC50 of 32 ± 1.4nM. D. Transcriptional activation of endogenous promoters by the basic synthetic cascade. The data are the mean ± SEM tdTomato expression for the mating (red), invasive growth (orange) and cell wall integrity (yellow) pathways normalized to background. E. Activation of ERK by endogenous stimuli. Data are the mean ± SEM phosphorylation level normalized to the estradiol stimulated cascade. See also Figure S1B.
Figure 2
Figure 2. The Variable Expression Cascades
A. Schematic of variable expression cascades. The kinase concentration increases at each tier in the cascade. B. Normalized fold increase of the high-MEK variable cascade (dark purple squares) and the basic cascade (black squares). The high-MEK variable cascade: single-copy Raf:ER expressed by PtetO7, high-copy MEK expressed by PtetO7, and high-copy ERK expressed by PGal (see also Figure S2). The data are the mean ± SEM, normalized to the fitted baselines. Data were fit with a modified Hill equation (solid lines). C. Model simulations of the high-MEK variable cascade (dark purple) and the basic cascade (black). The fitted concentrations were Raf:ER = 10nM, MEK = 80nM, and ERK = 100nM. D. Simulated Hill coefficient for the variable cascade in which both MEK and ERK were varied from 10nM to 100nM. E. Simulated EC50 for the variable cascade for the same parameters as in D. F. Normalized fold increase of the low-MEK variable cascade (light purple squares) and the basic cascade (black squares). The low-MEK variable cascade: single-copy Raf:ER expressed by PtetO7, single-copy MEK expressed by PGal, and high-copy ERK expressed by PGal. The data are the mean ± SEM, normalized to the fitted saturation line. Data were fit with a modified Hill equation (solid lines). G. Model simulations of the low-MEK variable cascade (light purple) and the basic cascade (black). The fitted concentrations were Raf:ER = 10nM, MEK = 30nM, and ERK = 100nM.
Figure 3
Figure 3. The Basic Cascade with Scaffolding
A. Schematic of the basic cascade co-expressed with a two-member scaffold pax*. See also Figure S3A. B. Percent response of the basic cascade alone and co-expressed with single-copy or high-copy pax*. The experimental data (red) are the mean ± SEM and the computational data (grey) are single values. Both are normalized to the maximum activation of the basic cascade in the absence of scaffold. C. Normalized fold increase of the basic cascade (black squares) co-expressed with single-copy (red squares) or high-copy (orange squares) pax*. The data are the mean ± SEM, normalized to the fitted baselines (basic and single-copy pax*) or the minimum and maximum values (high-copy pax*) for each condition. The data were fit with a modified Hill equation (black and red lines) or biphasic dose-response equation (orange line). D. A compartmental model of no (black), low (red) or high (orange) scaffold expression. Simulations are normalized to the maximum value of each condition. See also Figure S3B.
Figure 4
Figure 4. Negative Regulation of the Basic Cascade
A. Schematic of the basic cascade co-expressed with the ERK phosphatase MKP1-cyt. B. Normalized fold increase of the basic cascade (black squares) co-expressed with high-copy MKP1-cyt (green squares) expressed by PGal. The data are the mean ± SEM, normalized to the fitted baselines. The data were fit with a modified Hill equation (solid lines). C. Model simulations with (green) and without (black) MKP1-cyt co-expression. Data are normalized to the maximum value of each condition. D. Schematic of the basic cascade with the MEK inhibitor CI-1040. E. Normalized fold increase of the basic cascade (black squares) pre-treated with 50nM CI-1040 for 30 minutes prior to estradiol stimulation (blue squares). The data are the mean ± SEM, normalized to the fitted baselines. The data were fit with a modified Hill equation (solid lines). F. Model simulations with (blue) and without (black) inhibitor pre-treatment. Data are normalized to the maximum value of each condition.
Figure 5
Figure 5. Computational Analysis of Concentration Variation and Negative Regulation
A. No regulation: Raf:ER was held constant at 10nM, and MEK and ERK were varied from 10nM to 2μM and 10nM to 10μM, respectively. The top panel shows the Hill coefficient, the middle panel shows the EC50, and the bottom panel shows the normalized signal strength. On the top panel, the point marked “X” indicates the Xenopus cascade and the point marked “Y” indicates the yeast pheromone cascade. Each symbol represents a distinct class of system response (Table S1). For example, the square identifies systems for which the Hill coefficient is low, the EC50 is high and the normalized signal strength is low. On the bottom panel, the point marked “B” denotes the basic cascade and the point marked “V” denotes the high MEK variable cascade. See also Figures S4 and S5. B. MEK inhibition: The same parameter space as in A modeled with 50nM CI-1040. On the top panel, the star represents a distinct system response not seen in the absence of regulation. In the bottom panel, “B” indicates the basic cascade and “V” indicates the high-MEK variable cascade. C. ERK phosphatase: The same parameter space as in A modeled with 100nM MKP1-cyt co-expression. The star represents the system response in B achieved in a different region of parameter space.
Figure 6
Figure 6. Theoretical Analysis of Cascade-Generated Ultrasensitivity
A. Schematic of dual-step and single-step mass-action kinetic models and a lumped Hill equation model coupled to a dual-step tier. B. The Hill coefficients of each level of the cascade for both dual- and single-step mass-action kinetic models for representative low and high ultrasensitivity systems. See also Table S2. C. Simulations of distinct active MEK species for dual- and single-step cascades at the relative concentrations indicated in B. D. Comparison of simulated active MEK complex species with the Hill equation and mass-action kinetic models for the same input function (total active MEK) in the high ultrasensitivity system.
Figure 7
Figure 7. MEK Inhibition of the Variable Cascade
A. Normalized fold increase for the high-MEK variable cascade with (pink squares) and without (dark purple squares) pre-treatment with 50nM CI-1040 and for the basic cascade with (blue squares) and without (black squares) this inhibitor. The data are the mean ± SEM normalized to the fitted baselines for each condition. The data were fit with a modified Hill equation (solid lines). B. Model simulations of the experimental conditions described in A. C. Percent response of the basic cascade with (blue squares) and without (black squares) 50nM CI-1040 pre-treatment. The data are the mean ± SEM normalized to the maximum activation of the basic cascade in the absence of inhibitor. D. Model simulations of the experimental conditions described in C. E. Percent response of the high-MEK variable cascade with (pink squares) and without (black squares) 50nM CI-1040 pre-treatment. The data are the mean ± SEM normalized to the maximum activation of the high-MEK variable cascade in the absence of inhibitor. F. Model simulations of the experimental conditions described in E.
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