- Research article
- Open Access
Ultrasensitive Responses and Specificity in Cell Signaling
© Haney et al; licensee BioMed Central Ltd. 2010
- Received: 11 February 2010
- Accepted: 25 August 2010
- Published: 25 August 2010
Interconnected cell signaling pathways are able to efficiently and accurately transmit a multitude of different signals, despite an inherent potential for undesirable levels of cross-talk. To ensure that an appropriate response is produced, biological systems have evolved network-level mechanisms that insulate pathways from crosstalk and prevent 'leaking' or 'spillover' between pathways. Many signaling pathways have been shown to respond in an ultrasensitive (switch-like) fashion to graded input, and this behavior may influence specificity. The relationship of ultrasensitivity to signaling specificity has not been extensively explored.
We studied the behavior of simple mathematical models of signaling networks composed of two interconnected pathways that share an intermediate component, asking if the two pathways in the network could exhibit both output specificity (preferentially activate their own output) and input fidelity (preferentially respond to their own input). Previous results with weakly-activated pathways indicated that neither mutual specificity nor mutual fidelity were obtainable in the absence of an insulating mechanism, such as cross-pathway inhibition, combinatorial signaling or scaffolding/compartmentalization. Here we found that mutual specificity is obtainable for hyperbolic or ultrasensitive pathways, even in the absence of an insulating mechanism. However, mutual fidelity is impossible at steady-state, even if pathways are hyperbolic or ultrasensitive. Nevertheless, ultrasensitivity does provide advantages in attaining specificity and fidelity to networks that contain an insulating mechanism. For networks featuring cross-pathway inhibition or combinatorial signaling, ultrasensitive activation can increase specificity in a limited way, and can only be utilized by one of the two pathways. In contrast, for networks featuring scaffolding/compartmentalization, ultrasensitive activation of both pathways can dramatically improve network specificity.
There are constraints to obtaining performance objectives associated with signaling specificity; such constraints may have influenced the evolution of signal transduction networks. Notably, input fidelity (preferential response to an authentic input) is a more difficult objective to achieve than output specificity (preferential targeting to an authentic output). Indeed, mutual fidelity is impossible in the absence of an insulating mechanism, even if pathways are ultrasensitive. Ultrasensitivity does, however, significantly enhance the performance of several insulating mechanisms. In particular, the ultrasensitive activation of both pathways can provide substantial improvement to networks containing scaffolding/compartmentalization.
- Interconnected Network
- Mutual Specificity
- Output Specificity
- Basic Architecture
- Combinatorial Signaling
To survive, and to function as a part of a whole organism, cells must sense and respond both to their environment and to other cells. Cells sense a variety of chemical and physical signals, that are then transmitted and interpreted in a signal-specific fashion. These signals include hormones such as insulin and adrenaline, growth factors such as EGF (epidermal growth factor) and NGF (nerve growth factor), and physical signals such as mechanical stress, osmotic pressure, light, pH, etc.
For particular signals to elicit appropriate responses (e.g. turn on certain genes), the news that a signal has been detected must be accurately relayed to the intracellular machinery necessary to evoke the response (e.g. transcription factors). Signal transmission (also called signal transduction) is generally initiated by the activation of a cell surface or intracellular receptor. The receptor then typically activates a cascade of intracellular kinases, which then regulate various downstream effectors. It is commonplace, however, for these intracellular kinases to be involved in more than one signaling cascade. The need to respond to a multitude of different signals, combined with a high promiscuity of kinases, creates a complicated and interconnected network of signaling components [1–4]. This interconnectedness leads to the potential for crosstalk and cross regulation, where the signals from one pathway intersect with another. Cross regulation can be beneficial or even necessary when cells must integrate their response to multiple signals simultaneously . On the other hand, many signals necessitate a unique and decisive response, and a densely interconnected network may make such signal-exclusivity a difficult but vital objective to obtain [6–8]. Indeed, mutations that disrupt signaling specificity may play a role in the pathogenesis of cancer and other diseases [9, 10].
Issues of signaling specificity feature prominently in mitogen-activated protein kinase (MAPK) cascade-mediated signaling [10–16]. A textbook example is the mammalian Ras/MAPK cascade, as exemplified using rat PC12 cells as a model system. This cascade is a central component of the response to both EGF and NGF; however, EGF causes the cells to proliferate, whereas NGF causes them to differentiate and sprout neuron-like projections [15, 17–21]. Another textbook example is found in bakers/brewers yeast (Saccharomyces cerevisiae), where a set of overlapping MAPK cascades regulate mating, filamentous invasive growth, and stress-responses. In this case, elements of the same MAPK pathway are involved in transmitting at least three distinct signals: mating pheromone, nutrient limitation, and osmotic stress [13, 21]. Even so, there is specificity from signal to cellular response: application of pheromone elicits mating (but not filamentation or stress response), nutrient limitation elicits filamentation (but not mating or stress response), and osmotic stress elicits only a stress response.
How then do real signaling networks achieve specificity? Biochemical regulatory motifs knows as insulating mechanisms are thought to have evolved to maintain specificity by limiting 'leaking' or 'spillover' or 'bleed-through' between pathways. The fundamental insulating mechanism in cell regulation is specific protein-protein interactions [9, 26, 27], but this cannot account for specificity in networks containing pathways that share components. Insulating mechanisms that can buffer against spillover despite component sharing include combinatorial signaling, cross-pathway inhibition, compartmentalization, scaffolding, and kinetic insulation [22, 25, 28–30]. Some of the best experimental evidence for the existence and importance of these types of insulating mechanisms comes from the yeast MAPK network. In this system, multiple mutations that disrupt insulating mechanism function have been identified, and shown to result in increased levels of inappropriate signal crossover, often with adverse physiological consequences [29, 31–37]. Theoretical and modeling studies have supported the idea that insulating mechanisms can provide varying degrees of output specificity and input fidelity to interconnected signaling networks [22, 25, 38–42].
How do ultrasensitive (or hyperbolic) responses affect signaling specificity? Can ultrasensitivity provide signaling specificity to an interconnected network devoid of any insulating mechanisms? If not, does ultrasensitivity influence the performance of certain insulating mechanisms? These questions are challenging to address experimentally, so an approach using theory and modeling would seem a good starting point. Here we focus on simple phenomenological models of overlapping cascades that explicitly display various degrees of ultrasensitivity. The tractability of our approach allows us to derive analytic results that give a general insight into the effects of ultrasensitivity in achieving specificity. We find that ultrasensitivity cannot, by itself, provide specificity to an interconnected network, but that it can significantly enhance the performance of certain insulating mechanisms.
1. Definitions of Mutual Specificity and Mutual Fidelity
As in previous treatments, [22, 25], we consider a network consisting of two pathways, the X pathway and the Y pathway (Fig. 1A). Each pathway has a receptor/signaling component, x0 and y0, and a reporter/target component, x2 and y2. Notably, the two pathways share a common intermediate component, x1. Note that one component may be taken to represent the conglomeration of many chemical species. For example x0 may represent an entire G-protein-coupled receptor complex and several other steps upstream of a shared cascade x1. Hence, the network shown in Fig. 1A represents the simplest idealized "basic architecture" of a network in which two pathways share components. The input to each pathway will be given by specifying the levels of x0 and y0, and the output of each pathway will be measured by x2 and y2.
Let us denote the total output of pathway X when the cell is exposed to an × input signal (x0 > 0, y0 = 0) as Xout|Xin, read as 'X output given X input', or simply 'X given X'. In a similar fashion we define Y out |Y in , the value of y2 given that the Y pathway is activated (x0 = 0, y0 > 0). We also define the crosstalk terms X out |Y in (the value of x2 given that Y is activated) and Y out |X in . In this paper, we will use steady-state analysis so as to derive maximal analytical insight, so the outputs defined above refer to steady-state values.
where S X denotes the output specificity in the X pathway and F Y denotes the input fidelity in the Y pathway, etc.
MFMS greater than 1 indicates that each of S X , S Y , F X and F Y are simultaneously (meaning they are evaluated using the same parameters including input levels and connection strengths) greater than 1, and hence the cell signaling network faithfully communicates through both pathways. Note that these definitions may be readily generalized to include more than two intersecting signaling pathways.
In the rest of this paper we explore methods utilized by biological systems to obtain MFMS greater than 1.
2. A Model with Ultrasensitivity
where the Hill exponents, n and m, quantify the degree of ultrasensitivity. For hyperbolic pathways, the Hill exponent is equal to 1, whereas for ultrasensitive pathways, the Hill exponent is greater than 1. Indeed, the greater the Hill exponent, the more switch-like the response. For a Hill number of 1, it an 81-fold change in input to increase output from 10% to 90% maximal. In contrast, for Hill numbers of 2 and 4, it takes a 9-fold and 3-fold change, respectively.
3. Hyperbolic or ultrasensitive signaling pathways can achieve Mutual Specificity
Hence, it is clear that any effort to increase S X will result in a reciprocal decrease of S Y , so that both S X and S Y cannot be simultaneously greater than one. Neither mutual specificity nor mutual fidelity is possible with the basic architecture and weak activation; thus, some sort of insulating mechanism is required to obtain MFMS for weakly-activated pathways .
With a careful selection of parameters mutual specificity of any degree can be obtained (see Additional file 1 section 1a). For ultrasensitive pathways, we have already seen that mutual specificity can be obtained, since hyperbolic pathways are a sub-case of ultrasensitive pathways. So, while both hyperbolic pathways and ultrasensitive pathways can achieve mutual specificity of any degree, ultrasensitive pathways impose less stringent requirements on parameters. For a detailed discussion of the advantages provided by ultrasensitivity see Additional file 1 section 1b.
A pictorial representation of a pathway with no cross-regulation obtaining mutual specificity is given in Fig. 2A. To reiterate, mutual specificity is possible in networks containing hyperbolic or ultrasensitive pathways, even when the topology of such networks is simply the basic architecture without any added insulating mechanism. However, as we show next, it is still impossible to attain mutual fidelity without adding some kind of insulating mechanism to the basic architecture.
4. Mutual Fidelity cannot be obtained by the basic architecture
It should be noted that the specification that this be evaluated at steady state is crucial to this conclusion. There are certain conceivable ways to utilize a time-dependent signal to allow for mutual fidelity and mutual specificity with certain types of activation functions without imposing added regulation.
Note that if we had instead assumed that , then we would have concluded that fidelity in the Y pathway is guaranteed, whereas fidelity in the X pathway is impossible. Therefore, we cannot have both X and Y fidelity, i.e. mutual fidelity, regardless of the form of the monotonic activation functions, f X and f Y . In order to have mutual fidelity, one of the activation functions must be non-monotonic, that is, decreasing somewhere. This cannot be achieved by the basic architecture; it requires some type of added regulation.
5. Insulating mechanisms and cross-regulation
Biological signaling networks that share components are thought to contain one or more insulating mechanisms that provide specificity and fidelity [22, 25, 28–30]. From the analysis above it is clear that insulating mechanisms must be added if the basic architecture is to achieve mutual fidelity and mutual specificity. Here we briefly review three well-known insulating mechanisms, cross-pathway inhibition (CPI), combinatorial signaling (CS) and scaffolding/compartmentalization (SC) [22, 25] (Fig. 1. We will then develop the notion of a cross-regulatory term that facilitates the comparison of different insulating mechanisms. Then, in subsequent sections, we address the effects of ultrasensitivity on the performance of these insulating mechanisms.
Here production of y2 is inhibited by x2, with the amount of inhibition depending on the amount of x2. The parameter ε g is the IC50 (the inhibitory concentration 50%), which can be interpreted as the amount of x2 that results in 50% inhibition. When there is no x2, the production of y2 is unchanged; when x2 is much greater than ε g , y2 production is nearly completely shut off. Note that this insulating mechanism affects only the Y pathway's output and has no influence on X output. For a discussion of bi-directional mechanisms see Additional file 1 section 3.
Here, R[x0] represents the combinatorial input. As target x2 is a coincidence detector, its activity depends on two separate inputs, R and x1. If either input is zero, then x2 is also zero. When pathway X is on (and Y off), the coefficient R[x0] ≡ 1, and signal propagation through the network is identical to the basic architecture. When Y is on, R[x0] ≡ kleak, where kleak, a constant between zero and one, is the normalized basal level of signal flux from Z. Hence, X out |Y in will be reduced by a factor of kleak compared to the basic architecture. Hence, kleak = 1 has no specificity enhancing effect, whereas kleak = 0 completely eliminates X output given Y input. As with cross-pathway inhibition, combinatorial signaling only affects one output, in this case the X pathway output.
Signaling scaffolds are proteins that bind to two or more consecutively-acting components of a signaling cascade and, in so doing, facilitate signal transmission between them, (Fig. 1D). A prototypical example is the yeast Ste5 scaffold protein, which binds to all three tiers of the mating MAPK cascade . We refer to this as the sequestering function of scaffolds, to be distinguished from the selective activation function of scaffold proteins , which resembles combinatorial signaling .
SC becomes increasingly more effective as the exchange parameters D in , D out → 0. At this limit, the X and Y pathways have no crosstalk, and hence possess perfect (i.e. infinite) MFMS.
Cross Regulatory Term (CRT)
where for SC we let D in = D out ≡ D and .
Each of the CRTs were chosen intuitively as a set of parameters that quantifies the cross pathway regulation. For example with combinatorial signaling, the leak rate is clearly the parameter that quantifies the cross pathway regulation, as it is the only parameter that differentiates a CS network from the basic architecture. Both numerical (data not shown) and analytic results (see below) show that the CRTs as defined are in fact critical for determining specificity.
6. Ultrasensitivity can improve insulating mechanism performance
As we have seen, mutual fidelity at steady state is impossible without some kind of additional regulation. In this section we derive maximal values for MFMS for each of the insulating mechanisms, for networks with both linear and ultrasensitive activation. As stated above, in many cases one can show that arbitrarily high degrees of MFMS can be achieved at steady state. Here we derive bounds based on a fixed CRT. We also numerically evaluate the steady state values for each network at different levels of CRT to show that the bounds we derive are in fact sharp.
For linear activation, deriving expressions for each of the specificity indicators has been done previously . Here we shall re-formulate these in terms of the CRT.
Upon inspection F Y > 1 only when , which then makes F X < 1. Therefore, regardless of the CRT, MFMS ≤ 1.
The maximum of this expression over all of the parameters occurs when , and at this point .
Hence we obtain MFMS ≤ 1 + CRT.
Hence, in contrast to the case with weak-activation and cross-pathway inhibition, where mutual fidelity was impossible, when ultrasensitive, or even hyperbolic, activation is added to this architecture, MFMS > 1 can be obtained.
In both of these cases the degree to which ultrasensitivity helps is hidden. While the bounds for the hyperbolic(n = m = 1) and ultrasensitive case are the same, the speed at which they approach these bounds is much different. With high Hill exponents the constraints on the remaining parameters are much less stringent (see Additional file 1 section 1b). Further high degrees of ultrasensitivity can drastically decrease one of the crossterms X out |Y in or Y out |X in , see more on this in the next section.
In this case, unlike the cases of CPI or CS, the ultrasensitivity and CRT contributions to MFMS are intimately connected. This connection creates a super-linear increase in MFMS due to increasing CRT when ultrasensitivity is greater than one, in contrast with both CPI and CS where MFMS increases only linearly in CRT regardless of the degree of ultrasensitivity. This means that for networks with scaffolding/compartmentalization, even with a low value of the CRT, sufficient ultrasensitivity can serve to greatly increase MFMS, and visa versa. Note that in this symmetric case we have not derived a bound, the MFMS is in fact equal to this value. This is because the symmetric parameter choice greatly simplifies the situation by making S X = S Y = F X = F Y = MFMS.
Numerical evaluation of the specificity indicators confirm the bounds derived for the networks with ultrasensitive activation are also sharp (data not shown). Further numerical simulation shows that, in the case in which only symmetric parameters are used, as in (6.3), both the maxima and distribution of MFMS values are similar; so the results derived in this case should be representative of the more general case.
The bounds are plotted in Fig. 3 for comparison with those with linear activation. In each case the bounds with ultrasensitive activation clearly supercede those with linear activation. In particular, note the steep increase in MFMS due to the super-linear dependence on CRT in the case of scaffolding/compartmentalization.
In the case with scaffolding, high degrees of MFMS are achieved at relatively low levels of ultrasensitivity, n or m. For scaffolding with n = m = 10, MFMS is well over 100 (data not shown).
7. Strategies to maximize MFMS using ultrasensitivity
In this section we wish to understand why increasing only one Hill exponent is beneficial for CPI and CS, whereas increasing both is beneficial for scaffolding/compartmentalization as shown in section 6. To explain these observations we study how responsive the specificity and fidelity indicators are to changes in n and m by analyzing their partial derivatives.
which is a necessary and sufficient condition for both n and m to have positive effects on both S X and S Y . When this technique is applied to the fidelity indicators we derive the same conditions. Clearly, not all these conditions can be satisfied simultaneously, since either or .
After the addition of insulating mechanisms the exact derivatives change slightly, but Eqn. (7.2) still holds for both cross-pathway inhibition (CPI) and combinatorial signaling (CS). In other words adding either of these insulating mechanisms, or both simultaneously (see Additional file 1 section 3) will not change the fact that the equations in (7.1) cannot be simultaneously satisfied.
In CPI, both Y out |X in and Y out |Y in are decreased due to the inhibition by x2, which will allow for S X > 1 and potentially F Y > 1, but CPI does nothing to decrease the X out |Y in term. Thus in order to obtain fidelity in the × pathway, F X > 1, and hence MFMS > 1 it must be the case that:. Under this parameter choice, increasing n has only favorable effects, but increasing m has mixed effects; it increases S Y but decreases S X . In the case of CS the relation is the opposite because CS only effects the X out |Y in term and has no effect on the others. So the parameters must satisfy the relations: . Again if this were not the case we would not attain MFMS > 1, this time because F Y < 1. The consequence of this parameter choice, however, is that increasing n decreases S Y . In both cases the significance of not satisfying one of the above conditions is that increasing m, in the case of CPI, or n, in the case of CS, has detrimental effects on one of the specificity indicators, or increases one of the cross-terms X out |Y in or Y out |X in . The consequence of equation (7.2) is that networks with either CPI or CS can only utilize ultrasensitivity to decrease one of the crosstalk terms, X out |Y in or Y out |X in , at a time where the other cross-term must be kept small via cross-regulation. These beneficial effects of ultrasensitivity, however, greatly exceed those in linear or hyperbolic pathways or even those due to cross-regulation. The cross-terms that ultrasensitivity is able to decrease show polynomial decrease (and hence polynomial increase in corresponding specificity indicators) whereas the cross-terms that cross-regulation decrease, in the cases of CS and CPI, show only linear decrease (leading to a linear increase in corresponding specificity indicators). Thus due to the fact that MFMS is a minimum of the four specificity indicators (1.1), the bounds for CPI and CS show only linear increase with CRT.
This allows for the possibility of both n and m to increase MFMS. Therefore with scaffolding/compartmentalization, ultrasensitivity in both the X and Y pathways can simultaneously increase specificity.
Why does SC do so much better? Recall the issue in achieving mutual fidelity: if X out and Y out are activated in a monotone way mutual fidelity is impossible. Embellishing the system with insulation mechanisms is a way around this problem and hence insulation mechanisms are responsible for achieving MFMS. However in every case but SC, the problem remains that one of the x1 steady states must be lower than the other. For this reason there is no way to set the parameters so that increasing the ultrasensitivity simultaneously increases pathway specific variables, X out |X in and Y out |Y in , while decreasing the crosstalk terms, X out |Y in and Y out |X in . In SC, because the X pathway is only activated by and the Y pathway is only activated by one can set the threshold for input such that it is above the steady state level when given Y input but below the steady state level when given X input, and visa versa for the threshold. The consequences of this are that in this case ultrasensitivity can simultaneously decrease both cross-terms which allows for a polynomial decrease in both terms and thus a polynomial increase in MFMS as a whole, as seen in Figure 3C and equation (6.4). For example for n = 2 SC achieves MFMS at a level an order of magnitude higher than either CPI or CS.
Specificity in cell signaling pathways is often easy to observe. For instance, yeast cells mate when exposed to mating pheromone and form filaments when starved for nutrients. However this is an observation of whole cell behavior that either happens or not. Quantifying specificity is a more difficult task. Typically one measures the level of pathway specific outputs.
Specificity is defined here as a ratio of two different variables, X and Y, which represent the output of the X and Y pathways, respectively. When measuring this output from a real cell a common thing to measure would be a concentration of an activated kinase or the transcript of a pathway-specific gene; let us call this concentration of gene X. However when comparing this to the output from another pathway, one would be dividing concentration of gene X by the concentration of gene (or kinase) Y. But the concentration at which gene X triggers a physiological endpoint, like mating, may be very different, potentially by orders of magnitude, than the concentration at which gene Y triggers a different output.
To address this issue the two output variables must be normalized somehow so that the construct X out |X in is not given in units of concentration of a pathway-specific gene, but given in a unit-less percent of a characteristic concentration for this gene. Two reasonable choices for a characteristic concentration would be the basal level of activation of the gene under no input, or the maximal or steady state level of activation of the gene under its own input. Each choice of a characteristic level should be specific to the system being studied, and hence in the analysis above we assume that such a choice has already been made and the variables are fittingly normalized.
The choice of normalization can have mathematical consequences that lead to a reinterpretation of data. For this reason we discuss the consequences of normalizing by the steady state levels of activation (for a discussion of normalization using basal levels see ).
So in this case input fidelity in the X pathway is the same as output specificity in the Y pathway and visa versa. Further the idea of mutual specificity and mutual fidelity are one in the same.
How does this reduction effect the conclusions above? First, clearly mutual specificity is no longer possible without cross regulation, since mutual specificity and fidelity are now equated. The reason that this was possible before and is no longer possible is that attaining MS without cross regulation requires the maximal output of one of the pathways to become large, however with the new normalized species this is impossible as they are both bounded by one.
Secondly the bounds that we derived on MFMS, or more simply specificity in this case, still hold. In each case the degree of MFMS was limited by fidelity, see above. Recall that with no cross regulation mutual specificity of any degree is possible (see section 3) and in fact in this case it is possible to simultaneously maximize S X , S Y and F X but not F Y (see section 1a of the Additional file 1). Hence the reduction of the problem of achieving MFMS to achieving MF does not make the problem easier.
With a normalization such as this we get a great reduction in the equations to consider. Further the normalized construct makes some intuitive sense. The outputs are given in terms of percent of the activation that occurs when the appropriate input is given. So why not always express the outputs this way? The terms used to normalize the outputs, X out |Y in and Y out |Y in , are dependent on the input strength. So for a small input the variables are normalized to a smaller number (making them larger) whereas if a large input is used it is the opposite case. Also the input strength of each pathway can be independently varied. So it is possible to choose a strong input for the × pathway and a weak input for the Y pathway or visa versa. These choices could then alter the values of specificity in the system. Again the units of the input to each system is potentially different and hence comparing them is inappropriate. So we are again faced with the problem of normalizing the input based on some characteristic value.
The simple explanation to these issues is that the problem of normalization can be complicated and should be considered on a case-by-case basis. Here we assume that this has been done and all of the variables are unit-less.
Cell signaling is integral to numerous fundamental biological processes including development, mating, multicellularity, learning and memory, and many others. In addition, defects in cell-cell communication and signal transduction are central to the pathogenesis of many human diseases, such as cancer and diabetes. A quantitative understanding of the properties of cell signaling is of critical importance both for greater basic understanding and for the development of new clinical paradigms. A major obstacle to this goal, however, is the challenge of understanding the design logic underlying the complicated, interconnected networks in which most signaling pathways are embedded.
Here we have focused on mechanisms that provide specificity to interconnected networks containing distinct pathways that share components. We combined a framework for the analysis of specificity in cell signaling with simple mathematical models of interconnected networks. Using this approach, we examined how the stimulus-response properties of signaling pathways may influence their specificity. We compared weakly-activated pathways with hyperbolic or ultrasensitive pathways, asking if they could provide or enhance specificity, either alone or when combined with certain insulating mechanisms. We found that ultrasensitivity could not provide specificity on its own, but could enhance the performance of certain insulating mechanisms.
Ultrasensitivity can confer output specificity but not input fidelity
To measure specificity we focused on two metrics, mutual output specificity (MS), where both pathways preferentially activate their own outputs, and mutual input fidelity (MF), where both pathways preferentially respond to their own inputs. Examining the network denoted the "basic architecture", a generic, idealized network containing two pathways that share a component, we found that mutual fidelity is impossible at steady-state. Previously we showed that weakly-activated pathways can endow this architecture with neither mutual specificity nor mutual fidelity [22, 25]. Here, we significantly extended this finding by showing that, while both hyperbolic and ultrasensitive pathways can provide mutual specificity, neither can provide mutual fidelity. In fact, our analysis applies not only to hyperbolic and ultrasensitive pathways, but also to any monotonic stimulus-response (input-output) relationship, for reasons discussed below.
Why is input fidelity more difficult to obtain than output specificity? Consider the input-output functions for the X and Y pathways. When both input-output functions are straight lines (as they are when both pathways are weakly activated), they cannot cross; that is, they intersect at the origin but nowhere else. Therefore, it is impossible to choose a pair of input levels so that X output is greater than Y at the lower level of input, and Y output is greater than X at the higher level of input (or visa versa). In other words, mutual specificity is impossible. The input-output functions corresponding to hyperbolic and ultrasensitive pathways are curves, not straight lines. Hence, one can pick parameters such that these curves will cross at some point, and thence it will be possible to pick a pair of input levels on either side of the intersection point that will provide mutual specificity. Mutual fidelity, in contrast, can only be obtained if one of the input-output curves reaches a maximum and then bends back down (see Fig. 2); that is, this curve must be non-monotonic. Non-monotonic behavior in stimulus-response curves generally cannot be achieved by cascades of enzymes that exhibit standard kinetics (even if there is cooperativity or multisite phosphorylation); instead, some sort of negative feedback loop or cross-inhibition will be needed . In this sense, then, mutual input fidelity is more difficult to achieve, and also, perhaps, more difficult to evolve.
Another implication of this result is that, for interconnected networks to exhibit input fidelity (wherein pathways respond preferentially to authentic inputs), the basic network architecture must be embellished with an insulating mechanism(s). This insulating mechanism may work, for instance, by transforming one the stimulus-response curves into a non-monotonic function (e.g. cross-pathway inhibition), or it may act by splitting the stimulus (e.g. scaffolding/compartmentalization) or by splitting a single response curve into two (e.g. combinatorial signaling). Regardless, we should expect insulating mechanisms to be found wherever pathways share components yet exhibit specificity from signal to cellular response.
Ultrasensitivity dramatically improves the performance of insulating mechanisms
Although ultrasensitivity cannot provide mutual fidelity to the basic architecture by itself, it can significantly improve the performance of several different insulating mechanisms. To facilitate comparison, we defined a term denoted the Cross Regulatory Term (CRT) for each of the different insulating mechanisms. In every case this was a non-dimensional term that characterized the "strength" of the insulating mechanism. This allowed us to quantify the degree to which mutual specificity and mutual fidelity (MFMS) increased as a result of increases in the CRT. We found that, as the CRT was increased, there was a much sharper increase in MFMS in those networks featuring ultrasensitive activation than in those with linear (i.e. weak) or hyperbolic activation.
All the networks that we examined displayed sharper increases in MFMS with ultrasensitive activation than with linear or hyperbolic activation. However, for the networks that utilized combinatorial signaling or cross-pathway inhibition, it was not possible to utilize ultrasensitivity simultaneously in both pathways to the benefit of specificity. In other words, increasing the ultrasensitivity in one of the pathways was detrimental. In contrast, networks that combined scaffolding/compartmentalization with ultrasensitive activation could achieve very high levels of MFMS even at low levels of cross regulation, and ultrasensitivity in both pathways was beneficial.
To summarize, the hierarchy we have found is as follows: First, in the absence of an insulating mechanism and in the presence of linear activation, neither mutual specificity nor mutual fidelity is possible. Second, the addition of ultrasensitive activation allows for mutual specificity; in fact, with a careful selection of parameters, S X , S Y and F X can become unbounded. However, mutual fidelity still cannot be achieved. Third, the addition of cross-pathway inhibition or combinatorial signaling to an ultrasensitive system can achieve mutual fidelity, but with a linear dependence on the amount of cross-regulation. Finally, ultrasensitive systems utilizing scaffolding/compartmentalization can realize a super-linear increase in mutual specificity and mutual fidelity as the extent of cross-regulation is increased.
Constraints and opportunities
During evolution, as new signaling pathways emerged from the duplication and divergence of pre-exiting parts, the issue of specificity must have been paramount. Why component sharing is a widespread feature of cellular regulatory networks is a mystery. One possibility is that some low level of crosstalk between pathways is beneficial, but too much is bad; this would explain the existence of both crosstalk and insulating mechanisms. Another possibility is that duplication of part of a pathway, followed by the imposition of an insulating mechanism, is an easier evolutionary path to take than duplication of an entire pathway. Regardless, it seems possible that some of the constraints (e.g. input fidelity is hard to achieve) and opportunities (e.g. ultrasensitivity can help the performance of insulating mechanisms) identified here may have influenced the evolution of signal transduction networks.
See Additional file 1.
This work was supported by a National Institutes of Health/National Science Foundation joint initiative on Mathematical Biology through National Institute of General Medical Sciences grant GM75309 (L.B., Q.N.); by NIGMS Center grant GM76516 (L.B., Q.N.); and by NIGMS research grants GM60366, GM84332 and GM86883 (L.B.); and NSF grant DMS 0917492 (Q.N.). The authors would also like to thank an anonymous reviewer for a comment that lead to the inclusion of the section on normalization.
- Weng G, Bhalla US, Iyengar R: Complexity in biological signaling systems. Science. 1999, 284: 92-96. 10.1126/science.284.5411.92PubMed CentralView ArticlePubMedGoogle Scholar
- Schwartz MA, Baron V: Interactions between mitogenic stimuli, or, a thousand and one connections. Curr Opin Cell Biol. 1999, 11: 197-202. 10.1016/S0955-0674(99)80026-XView ArticlePubMedGoogle Scholar
- Papin JA, Hunter T, Palsson BO, Subramaniam S: Reconstruction of cellular signalling networks and analysis of their properties. Nat Rev Mol Cell Biol. 2005, 6: 99-111. 10.1038/nrm1570View ArticlePubMedGoogle Scholar
- Ubersax JA, Ferrell JE: Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol. 2007, 8: 530-541. 10.1038/nrm2203View ArticlePubMedGoogle Scholar
- Jordan JD, Landau EM, Iyengar R: Signaling networks: the origins of cellular multitasking. Cell. 2000, 2000: 193-200. 10.1016/S0092-8674(00)00112-4.View ArticleGoogle Scholar
- Skalhegg BS, Tasken K: Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci. 2000, 5: D678-693. 10.2741/SkalheggView ArticlePubMedGoogle Scholar
- Feng XH, Derynck R: Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005, 21: 659-693. 10.1146/annurev.cellbio.21.022404.142018View ArticlePubMedGoogle Scholar
- Laub MT, Goulian M: Specificity in two-component signal transduction pathways. Annu Rev Genet. 2007, 41: 121-145. 10.1146/annurev.genet.41.042007.170548View ArticlePubMedGoogle Scholar
- Pawson T: Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell. 2004, 116: 191-203. 10.1016/S0092-8674(03)01077-8View ArticlePubMedGoogle Scholar
- Shaul YD, Seger R: The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta. 2007, 1773: 1213-1226. 10.1016/j.bbamcr.2006.10.005View ArticlePubMedGoogle Scholar
- Schaeffer HJ, Weber MJ: Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol. 1999, 19: 2435-2444.PubMed CentralView ArticlePubMedGoogle Scholar
- Tan PB, Kim SK: Signaling specificity: the RTK/RAS/MAP kinase pathway in metazoans. Trends Genet. 1999, 15: 145-149. 10.1016/S0168-9525(99)01694-7View ArticlePubMedGoogle Scholar
- Schwartz MA, Madhani HD: Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae. Annu Rev Genet. 2004, 38: 725-748. 10.1146/annurev.genet.39.073003.112634View ArticlePubMedGoogle Scholar
- Qi M, Elion EA: MAP kinase pathways. J Cell Sci. 2005, 118: 3569-3572. 10.1242/jcs.02470View ArticlePubMedGoogle Scholar
- Murphy LO, Blenis J: MAPK signal specificity: the right place at the right time. Trends Biochem Sci. 2006, 31: 268-275. 10.1016/j.tibs.2006.03.009View ArticlePubMedGoogle Scholar
- Bardwell L: Mechanisms of MAPK signalling specificity. Biochem Soc Trans. 2006, 34: 837-841. 10.1042/BST0340837PubMed CentralView ArticlePubMedGoogle Scholar
- Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995, 80: 179-185. 10.1016/0092-8674(95)90401-8View ArticlePubMedGoogle Scholar
- Schamel WW, Dick TP: Signal transduction: specificity of growth factors explained by parallel distributed processing. Med Hypotheses. 1996, 47: 249-255. 10.1016/S0306-9877(96)90088-2View ArticlePubMedGoogle Scholar
- Gatti A: Divergence in the upstream signaling of nerve growth factor (NGF) and epidermal growth factor (EGF). Neuroreport. 2003, 14: 1031-1035. 10.1097/00001756-200305230-00025PubMedGoogle Scholar
- Santos SD, Verveer PJ, Bastiaens PI: Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate. Nat Cell Biol. 2007, 9: 324-330. 10.1038/ncb1543View ArticlePubMedGoogle Scholar
- Lodish H, Berk A, Kaiser CA, Krieger M, Scott MP, Bretscher A, Ploegh H, Matsudaira P: Moecular Cell Biology. 2007, New York: W. H. FreemanGoogle Scholar
- Komarova N, Zou X, Nie Q, Bardwell L: A Theoretical Framework for Specificity in Cell Signaling. Nature Molecular Systems Biology. 2005, 1: 2005.0023-Google Scholar
- Frey S, Millat T, Hohmann S, Wolkenhauer O: How quantitative measures unravel design principles in multi-stage phosphorylation cascades. J Theor Biol. 2008, 254: 27-36. 10.1016/j.jtbi.2008.04.037View ArticlePubMedGoogle Scholar
- Heinrich R, Neel BG, Rapoport T: Mathematical Models of Protein Kinase Signal Transduction. Molecular Cell. 2002, 9: 957-970. 10.1016/S1097-2765(02)00528-2View ArticlePubMedGoogle Scholar
- Bardwell L, Zou X, Nie Q, Kamorova N: Mathematical Models of Specificity in Cell Signaling. Biophys J. 2007, 92: 3425-3441. 10.1529/biophysj.106.090084PubMed CentralView ArticlePubMedGoogle Scholar
- Pawson T, Nash P: Protein-protein interactions define specificity in signal transduction. Genes Dev. 2000, 14: 1027-1047.PubMedGoogle Scholar
- Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, Laub MT: Rewiring the specificity of two-component signal transduction systems. Cell. 2008, 133: 1043-1054. 10.1016/j.cell.2008.04.040PubMed CentralView ArticlePubMedGoogle Scholar
- Whitmarsh AJ, Davis RJ: Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem Sci. 1998, 23: 481-485. 10.1016/S0968-0004(98)01309-7View ArticlePubMedGoogle Scholar
- Harris K, Lamson RE, Nelson B, Hughes TR, Marton MJ, Roberts CJ, Boone C, Pryciak PM: Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins. Curr Biol. 2001, 11: 1815-1824. 10.1016/S0960-9822(01)00567-XView ArticlePubMedGoogle Scholar
- Behar M, Dohlman HG, Elston TC: Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks. Proc Natl Acad Sci USA. 2007, 104: 16146-16151. 10.1073/pnas.0703894104PubMed CentralView ArticlePubMedGoogle Scholar
- O'Rourke SM, Herskowitz I: The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 1998, 12: 2874-2886. 10.1101/gad.12.18.2874PubMed CentralView ArticlePubMedGoogle Scholar
- Sabbagh W, Flatauer LJ, Bardwell AJ, Bardwell L: Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. Mol Cell. 2001, 8: 683-691. 10.1016/S1097-2765(01)00322-7PubMed CentralView ArticlePubMedGoogle Scholar
- Bao M, Schwartz M, Gantin G, Yates J, Madhani H: Pheromone-Dependent Destruction of the Tec1 Transcription Factor is Required for MAP Kinase Signaling Specificity in Yeast. Cell. 2004, 119: 991-1000. 10.1016/j.cell.2004.11.052View ArticlePubMedGoogle Scholar
- Chou S, Huang L, Liu HP: Fus3-Regulated Tec1 Degradation through SCFCdc4 Determines MAPK Signaling Specificity during Mating in Yeast. Cell. 2004, 119: 1-20. 10.1016/j.cell.2004.11.053View ArticleGoogle Scholar
- Flatauer L, Zadeh S, Bardwell L: Mitogen-Activated Protein Kinases with Distinct Requirements for Ste5 Scaffolding Influence Signaling Specificity in Saccharomyces cerevisiae. Molecular and Cellular Biolgy. 2005, 1793-1803. 10.1128/MCB.25.5.1793-1803.2005.Google Scholar
- Schwartz MA, Madhani HD: Control of MAPK signaling specificity by a conserved residue in the MEK-binding domain of the yeast scaffold protein Ste5. Curr Genet. 2006, 1-13.Google Scholar
- Yang HY, Tatebayashi K, Yamamoto K, Saito H: Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK. EMBO J. 2009, 28: 1380-1391. 10.1038/emboj.2009.104PubMed CentralView ArticlePubMedGoogle Scholar
- Somsen OJ, Siderius M, Bauer FF, Snoep JL, Westerhoff HV: Selectivity in overlapping MAP kinase cascades. J Theor Biol. 2002, 218: 343-354. 10.1006/jtbi.2002.3082View ArticlePubMedGoogle Scholar
- Schaber J, Kofahl B, Kowald A, Klipp E: A modeling approach to quantify dynamic crosstalk between the pheromone and the starvation pathway in baker's yeast. FEBS J. 2006, 273: 3520-3533. 10.1111/j.1742-4658.2006.05359.xView ArticlePubMedGoogle Scholar
- McClean M, Mody A, Broach JR, Ramanathan S: Cross-talk and decision making in MAP kinase pathways. Nature Genetics. 2007, 39: 409-414. 10.1038/ng1957View ArticlePubMedGoogle Scholar
- Zou X, Peng T, Pan Z: Modeling specificity in the yeast MAPK signaling networks. J Theor Biol. 2008, 250: 139-155. 10.1016/j.jtbi.2007.09.024View ArticlePubMedGoogle Scholar
- Hu B, Rappel WJ, Levine H: Mechanisms and constraints on yeast MAPK signaling specificity. Biophys J. 2009, 96: 4755-4763. 10.1016/j.bpj.2009.02.065PubMed CentralView ArticlePubMedGoogle Scholar
- Goldbeter A, Koshland DE: An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci USA. 1981, 78: 6840-6844. 10.1073/pnas.78.11.6840PubMed CentralView ArticlePubMedGoogle Scholar
- Ferrell JE: Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs. Biochem Sci. 1996, 21: 460-466. 10.1016/S0968-0004(96)20026-X.View ArticleGoogle Scholar
- Ferrell JE: Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. Curr Opin Cell Biol. 2002, 14: 140-148. 10.1016/S0955-0674(02)00314-9View ArticlePubMedGoogle Scholar
- Chou S, Huang L, Liu H: Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast. Cell. 2004, 119: 981-990. 10.1016/j.cell.2004.11.053View ArticlePubMedGoogle Scholar
- Bruckner S, Kohler T, Braus GH, Heise B, Bolte M, Mosch HU: Differential regulation of Tec1 by Fus3 and Kss1 confers signaling specificity in yeast development. Curr Genet. 2004, 46: 331-342. 10.1007/s00294-004-0545-1View ArticlePubMedGoogle Scholar
- Bao MZ, Schwartz MA, Cantin GT, Yates JR, Madhani HD: Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast. Cell. 2004, 119: 991-1000. 10.1016/j.cell.2004.11.052View ArticlePubMedGoogle Scholar
- Shock TR, Thompson J, Yates JR, Madhani HD: Hog1 mitogen-activated protein kinase (MAPK) interrupts signal transduction between the Kss1 MAPK and the Tec1 transcription factor to maintain pathway specificity. Eukaryot Cell. 2009, 8: 606-616. 10.1128/EC.00005-09PubMed CentralView ArticlePubMedGoogle Scholar
- Flatauer LJ, Zadeh SF, Bardwell L: Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae. Mol Cell Biol. 2005, 25: 1793-1803. 10.1128/MCB.25.5.1793-1803.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Angeli D, Ferrell JE, Sontag ED: Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems. Proc Natl Acad Sci USA. 2004, 101: 1822-1827. 10.1073/pnas.0308265100PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.