Ultrasensitization in a Phosphorylation–Dephosphorylation Cycle

<p>Stimulus-response of the phosphorylation–dephosphorylation cycle depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-g001" target="_blank">Figure 1</a>B for varying substrate expression levels, S_tot = S₀ + S₀K + S₁ + S₁P, on a double-logarithmic scale. The relative alterations in the response, S₁, for a given stimulus, K_tot = K + S₀K, elicited by a 5-fold change in substrate expression are indicated next to the vertical arrows. Parameters chosen: k_on,K = k_off,P = k_cat,P = 0.01; k_off,K = k_cat,K = 1; k_on,P = 1.6; P_tot = 1.25.</p

Ultrasensitization Due to Synexpression within a Signaling Cascade

<div><p>(A) Schematic representation of a signaling cascade subject to synexpression. An increase in the regulator, r, was assumed to result in a proportional increase in the expression of intermediates S and T, both of which are subject to covalent modification by (de)phosphorylation.</p><p>(B) Ultrasensitization due to synexpression within a signaling cascade measured numerically by plotting the normalized response as a function of the regulator concentration, r, where S_tot = T_tot = r (solid line). To show that synexpression enhances ultrasensitization, the case where the regulator, r, affects transcription of T only is also shown for S_tot = 10 (dashed line). Similar results were obtained for other values of S_tot or other stimulus strengths (data not shown). Parameters chosen: k_off,1 = k_off,5 = k_cat,2 = k_cat,6 = k_off,3 = k_off,7 = k_cat,8 = P_S,tot = P_T,tot = 1; k_on,1 = 0.02; k_on,5 = 0.2; k_on,3 = 2.1; k_on,7 = 2; k_cat,4 = 1.1; K_tot = 10.</p></div

Ultrasensitization Due to Substrate Sequestration

<p>The normalized maximal response of the phosphorylation–dephosphorylation cycle depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-g001" target="_blank">Figure 1</a>B is plotted as a function of substrate expression on a semilogarithmic scale for the limit of strong stimulation (according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-e001" target="_blank">Equation 1</a>), where K_tot >> P_tot. The threshold, S_tot,T, (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-e002" target="_blank">Equation 2</a>) was varied as indicated, while the Michaelis-Menten constant of the phosphatase, K_M,P, was kept constant and assumed to be unity. The scheme on the top indicates the mechanism of ultrasensitization: for weak substrate-expression, most of the substrate is sequestered on the enzyme–substrate complexes, S₀K and S₁P, while signal transmission via S₁ occurs as soon as substrate expression, S_tot, exceeds the threshold, S_tot,T.</p

Ultrasensitization Due to Activity Switching

<p>The normalized response of the phosphorylation–dephosphorylation cycle depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-g001" target="_blank">Figure 1</a>B is plotted as a function of substrate expression on a semilogarithmic scale for varying stimulus levels. To relate the plot to analytical results given in the main text (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-e004" target="_blank">Equation 4</a>), the stimulus, K_tot, is expressed as V_max,K and given in times of V_max,P. For the parameters chosen (k_on,K = 0.02; k_off,K = k_cat,K = k_off,P = 1; k_on,P = 2; k_cat,P = P_tot = 0.1), substrate sequestration is insignificant (i.e., <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-e003" target="_blank">Equation 3</a> does <i>not</i> hold) and the kinase is significantly less saturated than the phosphatase (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010054#pcbi-0010054-e005" target="_blank">Equation 5</a>). The scheme on the top indicates the mechanism of ultrasensitization: increasing substrate expression induces a switch from high overall phosphatase activity (S₁ → S₀) to high overall kinase activity (S₀ → S₁).</p

Slow and Fast Regulation of Cellular Signal Transduction

<div><p>(A) Schematic representation of cellular signal transduction. Upstream stimuli (e.g., hormones) result in altered gene expression by eliciting rapid intracellular responses such as transcription factors (fast regulation). The resulting changes in protein expression often in turn affect cellular signal processing of upstream inputs via transcriptional feedback or crosstalk (slow regulation).</p><p>(B) Schematic representation of a phosphorylation–dephosphorylation cycle, where the kinase K and the phosphatase P catalyze the (de)phosphorylation of the substrate, S. Hormonal stimulation (i.e., fast regulation) was modeled by altering the total kinase concentration, K_tot = K + S₀K, and the steady-state concentration of free phosphorylated substrate, S₁, was taken as the response. The impact of slow regulation was modeled by varying the concentration of the substrate (S_tot = S₀ + S₀K + S₁ + S₁P) or that of the phosphatase (P_tot = P + S₁P).</p></div

Validation of the top genes by TaqMan RT-PCR in the cell lines.

<p>For the complete results of RT-PCR measurements refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059503#pone.0059503.s006" target="_blank">Table S6</a>.</p

Overview of the study.

<p>Boxes with grey background represent training steps, while white background represents validation steps.</p

Circos plot of genes conferring multiple resistances.

<p>Circos plot of RT-PCR validated correlations for genes associated with resistance against multiple agents as identified by microarray analysis. The thickness of the ribbons correlate to the <i>log(p)</i> of the correlation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059503#pone-0059503-t002" target="_blank">Table 2</a>.). Note the high number of genes associated with sunitinib resistance and the single gene associated with lapatinib resistance. The two most informative genes are ANXA3 and RAB25, each associated with resistance against four agents.</p

Concentrations used in the cell lines to measure the resistance indexes.

<p>Concentrations used in the cell lines to measure the resistance indexes.</p

Survival plots.

<p>Kaplan-Meier survival plots of sunitinib-treated metastatic RCC samples divided into two cohorts based on the median of EpCAM positive cells (p = 0.01).</p