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1 IUBMB Life, 58(11): , November 2006 Research Communication Sequestration Shapes the Response of Signal Transduction Cascades Nils Blu thgen Molecular Neurobiology, Free University Berlin, and Institute for Theoretical Biology, Humboldt University Berlin, Berlin, Germany Summary Many signal transduction cascades are composed of covalent modification cycles such as kinase/phosphatase cycles. In the 1980s Goldbeter and Koshland showed that such cycles can exhibit nonlinear input-output relations when the enzymes are saturated by their substrates, which may facilitate signal processing. Recent papers show that this mechanism is unlikely to cause non-linearity in mammalian signal transduction cascades as sequestration of the target due to enzyme concentrations present in these cascades will hamper this mechanism. However, sequestration due to high-affinity enzymes can shape the dynamics and steady-state behaviour of signal transduction cascades in different ways, some of which are discussed in this review. IUBMB Life, 58: , 2006 Keywords Signal transduction cascades; covalent-modification cycles; sequestration INTRODUCTION The dominating biochemical building blocks of signal transduction pathways in eukaryotes are covalent-modification cycles. In these cycles, a target molecule is modified, for example phosphorylated, and thereby activated. The modification is reversed usually by phosphatases that remove the phosphate (see Fig. 1A). Thereby the amount of phosphorylated target represents a balance between activating kinases and deactivating phosphatases. Parallels between information processing in signal transduction cascades and in neuronal networks have been pointed out (1). Similarly as in neuronal networks, signal transduction cascades make their decisions in parallel, interacting cascades Received 4 August 2006; accepted 5 September 2006 Address correspondence to: Nils Blu thgen, Molecular Neurobiology, Free University Berlin, Takustr. 6, Berlin, Germany. Tel: þ Fax: þ nils@itb.biologie.hu-berlin.de based on a multitude of inputs. In this analogy each covalent modification cycle corresponds to a neuron. Importantly, signal processing in neuronal networks requires highly nonlinear input-output relations in neurons such as thresholds for action potentials to perform their computation. Even though the topology of a intra-cellular signal transduction network is different from a neuronal network, nonlinear input-output relations are needed to convert gradients of inputs into binary decisions, e.g., during pattern formation in development (2). Therefore, input-output relations of covalent modification cycles are expected to show non-linearities to facilitate signal processing. In the early 1980s, Goldbeter and Koshland wrote a couple of landmark papers in which they showed that a covalent-modification cycle can react extremely non-linearly on the ratio of kinase and phosphatase activity (3 5). The necessary condition to obtain a highly nonlinear input-output relation is that the total substrate concentration exceeds the kinase s and phosphatase s K M -value, so that they can be saturated by their, substrate. In saturation, the reaction rates become virtually independent of the substrate concentration. At low kinase concentrations, most target is unphosphorylated and the kinase is saturated. At increasing kinase concentration more target is phosphorylated and leads to phosphatase saturation. Then the dephosphorylation rate is limiting and thus the modification state of the target flips abruptly from a rather unphosphorylated state to a rather phosphorylated state (3, the mechanism is nicely reviewed in Ref. 6 and 7). Goldbeter and Koshland have termed this phenomenon zero-order ultrasensitivity to emphasize that the enzymes need to be operating in their zeroth order, i.e., substratesaturated, regime. This behavior has been found in several metabolic systems such as in the phosphorylation of isocitrate dehydrogenase (8) and muscle glycolysis (9). However, it remains unclear whether this mechanism operates also in signal transduction pathways to facilitate non-linear signal processing, although a recent paper suggests this but did not prove it (10). These days, more and more data about protein abundance accumulates and suggests that enzymes and substrates in ISSN print/issn online Ó 2006 IUBMB DOI: /

2 660 BLU THGEN covalent modification cycles in mammalian signal transduction cascades are present in similar abundance. Then, the enzymes can bind to their targets preventing other enzymes from binding to the same docking site. Thereby enzymes can sequester the target from the pool of accessible targets. Having this in mind we and others have recently revisited the theoretical analysis of covalent modification cycles, and the results shed new light on the operation of these cycles. In this review, first it is briefly discussed why zero-order ultrasensitivity is in general unlikely to appear in signal

3 ROLE OF SEQUESTRATION IN SIGNAL TRANSDUCTION CASCADES 661 transduction pathways. Then other emergent properties of covalent modification cycles that appear at high enzyme concentrations are reviewed. ZERO ORDER ULTRASENSITIVITY REVISITED A necessary condition for zero-order ultrasensitivity to appear is that the enzymes can be saturated, which means that a large fraction of the enzyme is bound to its substrate. In turn, the substrate that is bound by the enzyme is not available to other enzymes and thus sequestered (see Fig. 1B). If the substrate is present in similar concentration as the enzymes also a substantial portion of the substrate is sequestered. By means of metabolic control analysis and dynamical systems theory, we and Salazar & Ho fer showed independently that substrate sequestration conflicts with the appearance of zero-order ultrasensitivity (11, 12). Under most circumstances, the substrate is sequestered by the enzymes such that the amount of free substrate drops below the enzyme s K M -values. Consequently, the enzymes are not saturated anymore and the cycle s stimulus-response curve approaches a hyperbolic response curve (see Fig. 1C). Under some special conditions there might be still some thresholdlike activation, however the substrate is sequestered to such an extend that essentially no free activated target remains to further signal downstream. Goldbeter and Koshland already observed that non-negligible enzyme-substrate complexes can change ultrasensitivity drastically. They argue that the sequestered target might still be catalytically active, if docking-site, modification site and catalytic site are well separated (3). We could, however, show that the response of both the free and sequestered target combined is necessarily less steep than the response of the free target alone. Thus, the attenuation of sensitivity by sequestration cannot be restored by an active phosphatase-target complex (12). The players signaling pathways have often multiple functions: they are substrates in covalent modification cycles and the modified substrate is an enzyme in the next cycle. Therefore they might be sequestered by molecules distinct from their own modifying enzymes. This causes additional sequestration which reduces the free substrate and thus limits zero-order ultrasensitization further (12). Other studies have also shown that product-sensitivity of the enzymes additionally hampers zero-order ultrasensitivity (13). Taken together, zero-order is an unlikely candidate for causing non-linearity in signal transduction and processing in higher eukaryotes. The importance of zero-order ultrasensitivity in metabolic regulation, however, is untouched by these results and remains high, as in typical metabolic systems the substrate concentration exceeds the enzyme concentrations by orders of magnitude. The fact that the kinases and phosphatases may sequester significant amounts of its target may yield other interesting effects in signal transduction cascades. These shall be discussed in the following. ULTRASENSITIZATION Legewie et al. have looked into a different input-output relation: how is the activity of a target in a covalent modification cycle controlled by its expression level (compare Fig. 1D) (14). Investigating this relation is interesting as relatively mild fold-changes in expression of several genes result in large effects. For example, several tumour-suppressor genes do not follow Knudson s two-hit hypothesis: even a loss of a single allele corresponding to a loss in expression by factor two is sufficient to abrogate their functionality. Interestingly, many of these tumour suppressors are phospho-proteins and are therefore substrate in covalent modification cycles, e.g., p53, BRAC1/2, H2AX, and prb. Legewie and colleagues showed by mathematical modelling that phosphatases with typical K M -values may sequester the target as long as it is expressed at levels below the phosphatase concentration (situation 1 in Fig. 1E and F ). Then it is not accessible by the kinase and can therefore not be activated. As soon as the target concentration exceeds the phosphatase concentration (plus a factor that corrects for different catalytic rates of phosphatases and kinases) the target can be phosphorylated (situation 2 in Fig. 1E and F ). Thereby covalent 3 Figure 1. Effects of sequestration in covalent modification cycles. (A) A typical covalent modification cycle: a target T is phosphorylated by the stimulating kinase and dephosphorylated by the opposing phosphatase. The phosphorylated target is active and signals downstream. (B) Phosphatase Pase and kinase K bind to the target T and thereby sequester it. (C) At low enzyme concentrations the covalent modification cycles might exhibit a sigmoidal input-output relation (red), this behavior disappears at enzyme concentrations similar to the target concentration, then the stimulus-response curve approaches a hyperbolic relation (D F). The amount of activated target can depend nonlinearly on the expression of the target. In situation 1, most of the target is sequestered by the phosphatase. If the expression level rises above the concentration of the phosphatase, it cannot be further sequestered and is activated (situation 2). (G) The target can be sequestered and deactivated by the phosphatase in a different compartment. H þ I: Sequestration of the kinase can delay double-phosphorylation. First, the kinase is sequestered to the unphosphorylated target (situation 1). Production of the mono-phosphorylated form relieves the kinase, and allows for modification of the second site (situation 2). The blue time-course shows the situation without any sequestration, the red time-course shows the situation for kinase sequestration. Notably, deactivation after removal of the stimulus (gray line) does not differ between both cases.

4 662 BLU THGEN modification cycles may suppress the activity of a gene expressed below a threshold. The authors called this effect ultrasensitization. Interestingly, sequestration by phosphatases has been suggested to occur in the classical MAPK pathway, where nuclear phosphatases may deactivate and anchor MAPK in the nucleus (15). Unlike in the model by Legewie et al. the phosphatases dephosphorylate and sequester only when its target is translocated to the nucleus, i.e., after being phosphorylated (see Fig. 1G). Thus sequestration by phosphatases in different compartments may cause different effects: Ultrasensitization if kinase and phosphatase operate in the same compartment, and signal termination if they operate in different compartments. MULTISITE PHOSPHORYLATION A large fraction of proteins are reversibly covalently modified at several sites. As early as in 1976, where only the phosphorylation of five proteins has been studied in detail, it was realized that three of them possess multiple phosphorylation sites (16). If all phosphorylation sites are being modified by the same kinase, the differently phosphorylated forms do all compete for this kinase. Then interesting dynamical effects may occur due to kinase sequestration. If most protein is unphosphorylated, the kinase will be recruited by the unphosphorylated form and sequester it away from the monophosphorylated form. This way it delays the second phosphorylation (see Fig. 1H and I). This delay, however, is sign-sensitive, that is it delays phosphorylation but not dephosphorylation. Such sign-sensitive delays have been suggested to play an important role in signal processing as they filter out short activations that might occur by sheer chance (17). Similar dynamical effects may occur due to shared phosphatases even when the kinase phosphorylating the sites is distinct. Bistability is another interesting effect that can be caused by sequestration of shared kinases and phosphatases in cycles where multiple sites are phosphorylated. It is interesting to note that here the sensitivity is increased due to sequestration effects, in contrast to zero-order ultrasensitivity, where sensitivity is weakened (18). Multisite-phosphorylation is an example of sharing an enzyme for several sites. If multiple phospho-proteins share a phosphatase or kinase, other interesting effects can occur due to sequestrations, as discussed in the following. CROSSTALK AND COMPETITION BY SHARING AN ENZYME OR SUBSTRATE Several phosphorylation-dephosphorylation cycles share their phosphatases. Thus, if one cycle is activated, it recruits the phosphatase and sequesters it away from the other cycle. As the amount of phosphatase for the other cycle is lowered it may respond to lower stimuli. Thereby two pathways can cross-talk with each other if their covalent modification cycles share a phosphatase. Then, if one pathway is activated, the threshold of the other is lowered as its phosphatase is sequestered away. In a recent paper Legewie and colleagues investigated the effect of such shared deactivators or inhibitors in cycles that are members of the same cascade (19). These cascades may exhibit bistability accompanied by hysteresis, i.e., when a threshold is reached, the cascade is rapidly activated and once activated it remains activated even if the stimulus is lowered to a certain degree. If the stimulus drops below a second (lower) threshold, the cascade can be deactivated. In contrast, shared activators may show a completely different effect. In the JAK/STAT pathways Janus Kinases (JAKs) are shared by several receptors. Dondi and colleagues showed experimentally that over-expression of one receptor can sequester JAKs and thereby reduce the activity of other receptors (20). CONCLUSIONS It is very likely that phosphatases and kinases with high affinity to their substrate cause non-linearity and thereby facilitate signal processing. The mechanism by which they achieve it is, however, in general unlikely to be zero-order ultrasensitivity. In this review I have argued that it is more likely to be through different sequestration effects: Causing thresholdlike activation for different expression levels via a mechanism called ultrasensitization, influencing the sub-cellular location by sequestering the target in the nucleus or by sequestering the predominant form in multisite-phosphorylation. Sequestration may also cause crosstalk between different cascades or bistability when a phosphatase is shared within a cascade. It is surprising to see that even modules as simple as covalent modification cycles can display very different kinds of dynamics and are neither experimentally nor theoretically fully understood (7). It is likely that we are just at the beginning to understanding how these cycles facilitate signal transduction cascades to compute adaptive responses to the signals available at the receptor level. ACKNOWLEDGEMENTS I thank Stefan Legewie, Frank Bruggeman and Hanspeter Herzel for stimulating and exciting discussions. This work is supported from BMBF through the Berlin Bernstein Center for Computational Neuroscience and from DFG through SFB 618. REFERENCES 1. Bray, D. (1995) Protein molecules as computational elements in living cells Nature 376, Lewis, J., Slack, J., and Wolpert, L. (1977) Thresholds in development. J. Theor. Biol. 65,

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