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Ultrasensitivity and Positive Feedback to Promote Sharp Mitotic Entry
Molecular Cell
Previews Ultrasensitivity and Positive Feedback to Promote Sharp Mitotic Entry Youlian Goulev1 and Gilles Charvin1,* 1Institut
de Biologie Mole´culaire et Cellulaire, 1 Rue Laurent Fries, 67400 Illkirch Cedex, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.01.016
In this issue, Trunnell et al. (2011) show that in mitotic entry the positive feedback that drives the activation of cyclin-dependent kinase (Cdk) involves a very ultrasensitive step of phosphorylation of Cdc25C by Cdk, thus strongly contributing to the switch-like behavior of this essential cell-cycle transition. Entry into mitosis is an essential event of the eukaryotic cell cycle, which is triggered by the progressive accumulation of mitotic cyclin B1 during the G2 phase. A key feature of this cell-cycle transition is that a rather gradual increase in the level of the trigger results in a sharp, robust and hysteretic activation of the cyclin B1-Cdk complex, which drives the progression of the mitotic machinery (Pomerening et al., 2003; Sha et al., 2003). Positive feedback in the control of cyclin B1-CDK activity by the two regulators Wee1 and Cdc25C has long been proposed to account for the emergence of a bistable transition (Novak and Tyson, 1993; Thron, 1996), yet a detailed quantitative and mechanistic analysis of the contribution of these feedback loops was largely missing. Recently, using X. laevis egg extracts, Kim and Ferrell quantified the inactivation of Wee1 by the cyclin B1-Cdk complex and provided new insights regarding the mechanism underlying the observed ultrasensitivity (Kim and Ferrell, 2007). In this issue of Molecular Cell, Trunnell and colleagues use the same methodology to focus on the activation of Cdc25C by the cyclin B1-Cdk complex. Strikingly, their results show that the phosphorylation of Cdc25C by cyclin B1-Cdk is a strongly ultrasensitive process that originates, to some extent, from multisite phosphorylation. Then they show how a simple mathematical model that integrates the respective contributions of the Wee1 and Cdc25 feedback loops can quantitatively account for the bistable switch observed in mitotic entry. The cell-cycle progression is driven by an autonomous biochemical clock that functions as a negative feedback oscil-
lator: the progressive rise in cyclin B11 level during G2 leads to the formation of a cyclin B1-Cdk complex, which indirectly triggers cyclin proteolysis by enabling the anaphase promoting complex/cyclosome (APC/C). Cyclin’s destruction in late mitosis then leads to low cyclin B1-Cdk activity, thus permitting exit from mitosis and also resetting the system for the next cycle. The activation of cyclin B1-Cdk kinase function requires its dephosphrylation at Tyr15 (and Thr14 in animal cells), which is a reversible process: dephosphorylation is promoted by the Cdc25 phosphatase, but this reaction is reversed by the Wee1 (and Myt1) kinases (see Figure 1A). Most interestingly, the activities of both Wee1 and Cdc25 are themselves regulated by active cyclin B1Cdk, thus providing an additional layer of feedback control on Cdk activity: Wee1 (resp. Cdc25) is inhibited (resp. activated) by cyclin B1-Cdk. As a result, the regulation of cyclin B1-Cdk activity features two mirror-image positive feedback loops. Positive feedback can induce bistability in cyclin B1-Cdk activity, a phenomenon that has several striking properties. First, within a given interval of level of the cyclin B1 trigger, it can either be in a low or a high Cdk activity state, but it cannot be stable at intermediate levels. Then, when the switch from low to high activity is triggered by increasing levels of cyclin B1, the discontinuity between the two stable states necessarily makes the transition extremely abrupt. Last, the transitions from the low to the high state and from the high to the low state occur for different level of the trigger (i.e., different levels of cyclin B1), a phenomenon known
as hysteresis. Bistability in cyclin B1-Cdk activity was predicted to occur in mitotic entry by early theoretical work (Novak and Tyson, 1993; Thron, 1996) and was indeed later observed in Xenopus egg extracts (Pomerening et al., 2003; Sha et al., 2003). Recently, it was shown to have strong functional implications by increasing the robustness of oscillations in Cdk activity during the cell cycle (Pomerening et al., 2005). Yet, positive feedback does not make a system necessarily bistable. The emergence of this property depends on the detailed architecture of the regulatory network and is also very much influenced by the values of its component parameters, such as the sensitivity of its feedback loops. In order to connect the architecture of a regulatory module to its systems-level behavior, it is therefore crucial to measure the individual response of its subparts in a quantitative manner. In the present issue of Molecular Cell, Trunnell et al. (2011) use Xenopus egg extracts to quantify the response of Cdc25C hyperphosphorylation (which is required for its activation) to variable cyclin B1-Cdk1 activity, in order to evaluate the sensitivity of the Cdc25C feedback loop. To isolate this reaction from the rest of the cell-cycle machinery, the authors use a recombinant mutant of Cdk1 (Cdk1AF) that cannot be inactivated by Wee1 and does not need to be activated by Cdc25C but is still able to phosphorylate Cdc25C. By varying cyclin B1-Cdk1 activity with added nondegradable cyclin B1 (D65-cyclin B1), they show that the Cdc25C hyperphosphorylation is a highly ultrasensitive function of Cdk1 activity (i.e., it displays a sharp sigmoidal behavior), as revealed by the
Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc. 243
Molecular Cell
Previews
exceptionally high value The model also provides A Cdc25 (around 11) measured for a useful framework in which cyclin B1 the Hill coefficient. In addito test the functional imUltrasensitivity tion, the dephosphorylation portance of specific features Activation step of Ser 287 in Cdc25C of this regulatory module: cyclin B1 Cdk1 Cdk1 Cdk1 Mitotic entry cyclin B1 (which is also involved in for instance, it clearly shows Inactivation Cdc25C activation) displays that high ultrasensitivity in P Ultrasensitivity an even higher Hill coefficient Cdc25C phosphorylation of about 32. strongly contributes to make Wee1 Interestingly, Trunnell and the Cdk activation system B colleagues find that the ultrabistable (see gray plots on sensitivity in Cdc25C hyperFigure 1B), Most importantly, 100 phosphorylation can only be it reveals that, while only one partially recapitulated with a of the two positive feedback 80 more simple in vitro system. loops is enough to induce This reveals that, similarly as bistability, the presence of 60 in the Wee1 regulation loop both regulatory elements pro(Kim and Ferrell, 2007), vides a larger robustness of extrinsic factors in the exthis property to parameter 40 tracts—that yet remain to be modifications, and therefore n=24 n=11 determined—are likely to may buffer against evolutionn=6 20 n=3 n=1 enhance the nonlinearity in driven changes. the Cdc25C phosphorylation In summary, this study thus 0 process. Based on further further contributes to bridge 0 20 40 60 80 100 observations showing that the gap between the molecTotal cyclin B1 (nM) this nonlinearity can be comular properties of Cdk activapletely suppressed by mutation and the systems-level Figure 1. Ultrasensitivity and Bistable Response in the Activation of Cyclin B-Cdk tion of three out of the consequences on this canon(A) Sketch of the regulation of the activation of the cyclin B-Cdk complex, numerous known phosphorical cell-cycle transition. which involves reciprocal positive (Cdc25C) and double-negative (Wee1) ylations sites in Cdc25C, the feedback loops. authors conclude that the (B) Role of Cdc25 ultrasensitivity in promoting bistable transitions. The steadyREFERENCES state concentrations of active cyclin B1/Cdk1 as a function of cyclin B1 are observed in vitro ultrasensiplotted according to the kinetic model described in Trunnell et al. (2011). tivity arises from multisite Kim, S.Y., and Ferrell, J.E., Jr. (2007). Each gray plot corresponds to the steady-state solution for a given value of Cell 128, 1133–1145. phosphorylation, and three the Cdc25C Hill coefficient, as indicated on the graph. The red curve (nH = 11) and corresponding arrows display the low-to-high and high-to-low transidifferent plausible mechaNovak, B., and Tyson, J.J. (1993). tions in cyclin B-CDK activity as cyclin B is varied (hysteresis). nisms are proposed to J. Cell Sci. 106, 1153–1168. account for this observation. Pomerening, J.R., Sontag, E.D., and What may be the implications of such a very good quantitative agreement with Ferrell, J.E., Jr. (2003). Nat. Cell Biol. 5, 346–351. ultrasensitivity on the function of the bi- the previously reported hysteretic stable mitotic trigger? To answer this behavior for the steady-state activity of Pomerening, J.R., Kim, S.Y., and Ferrell, J.E., Jr. (2005). Cell 122, 565–578. question, Trunnell et al. turn to a kinetic cyclin B1-Cdk as a function of cyclin B1 model of activation of cyclin B-Cdk that level (Pomerening et al., 2003). It therefore Sha, W., Moore, J., Chen, K., Lassaletta, A.D., Yi, integrates the respective contributions of strongly suggests that cyclin B1-Cdk C.S., Tyson, J.J., and Sible, J.C. (2003). Proc. Natl. Acad. Sci. USA 100, 975–980. the Wee1 and Cdc25 feedback loops. switch-like activation upon mitotic entry This simple mathematical analysis, al- (and inactivation upon mitotic exit) are Thron, C.D. (1996). Biophys. Chem. 57, 239–251. most entirely constrained by experimen- such as to follow a complete hysteresis Trunnell, N.B., Poon, A.C., Kim, S.Y., and Ferrell, J.E., Jr. (2011). Mol. Cell 43, this issue, 263–274. tally determined parameters, displays cycle, as shown in Figure 1B (red curve). Steady-state active cyclin B1/Cdk1 (nM)
*
244 Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc.
Previews Ultrasensitivity and Positive Feedback to Promote Sharp Mitotic Entry Youlian Goulev1 and Gilles Charvin1,* 1Institut
de Biologie Mole´culaire et Cellulaire, 1 Rue Laurent Fries, 67400 Illkirch Cedex, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.01.016
In this issue, Trunnell et al. (2011) show that in mitotic entry the positive feedback that drives the activation of cyclin-dependent kinase (Cdk) involves a very ultrasensitive step of phosphorylation of Cdc25C by Cdk, thus strongly contributing to the switch-like behavior of this essential cell-cycle transition. Entry into mitosis is an essential event of the eukaryotic cell cycle, which is triggered by the progressive accumulation of mitotic cyclin B1 during the G2 phase. A key feature of this cell-cycle transition is that a rather gradual increase in the level of the trigger results in a sharp, robust and hysteretic activation of the cyclin B1-Cdk complex, which drives the progression of the mitotic machinery (Pomerening et al., 2003; Sha et al., 2003). Positive feedback in the control of cyclin B1-CDK activity by the two regulators Wee1 and Cdc25C has long been proposed to account for the emergence of a bistable transition (Novak and Tyson, 1993; Thron, 1996), yet a detailed quantitative and mechanistic analysis of the contribution of these feedback loops was largely missing. Recently, using X. laevis egg extracts, Kim and Ferrell quantified the inactivation of Wee1 by the cyclin B1-Cdk complex and provided new insights regarding the mechanism underlying the observed ultrasensitivity (Kim and Ferrell, 2007). In this issue of Molecular Cell, Trunnell and colleagues use the same methodology to focus on the activation of Cdc25C by the cyclin B1-Cdk complex. Strikingly, their results show that the phosphorylation of Cdc25C by cyclin B1-Cdk is a strongly ultrasensitive process that originates, to some extent, from multisite phosphorylation. Then they show how a simple mathematical model that integrates the respective contributions of the Wee1 and Cdc25 feedback loops can quantitatively account for the bistable switch observed in mitotic entry. The cell-cycle progression is driven by an autonomous biochemical clock that functions as a negative feedback oscil-
lator: the progressive rise in cyclin B11 level during G2 leads to the formation of a cyclin B1-Cdk complex, which indirectly triggers cyclin proteolysis by enabling the anaphase promoting complex/cyclosome (APC/C). Cyclin’s destruction in late mitosis then leads to low cyclin B1-Cdk activity, thus permitting exit from mitosis and also resetting the system for the next cycle. The activation of cyclin B1-Cdk kinase function requires its dephosphrylation at Tyr15 (and Thr14 in animal cells), which is a reversible process: dephosphorylation is promoted by the Cdc25 phosphatase, but this reaction is reversed by the Wee1 (and Myt1) kinases (see Figure 1A). Most interestingly, the activities of both Wee1 and Cdc25 are themselves regulated by active cyclin B1Cdk, thus providing an additional layer of feedback control on Cdk activity: Wee1 (resp. Cdc25) is inhibited (resp. activated) by cyclin B1-Cdk. As a result, the regulation of cyclin B1-Cdk activity features two mirror-image positive feedback loops. Positive feedback can induce bistability in cyclin B1-Cdk activity, a phenomenon that has several striking properties. First, within a given interval of level of the cyclin B1 trigger, it can either be in a low or a high Cdk activity state, but it cannot be stable at intermediate levels. Then, when the switch from low to high activity is triggered by increasing levels of cyclin B1, the discontinuity between the two stable states necessarily makes the transition extremely abrupt. Last, the transitions from the low to the high state and from the high to the low state occur for different level of the trigger (i.e., different levels of cyclin B1), a phenomenon known
as hysteresis. Bistability in cyclin B1-Cdk activity was predicted to occur in mitotic entry by early theoretical work (Novak and Tyson, 1993; Thron, 1996) and was indeed later observed in Xenopus egg extracts (Pomerening et al., 2003; Sha et al., 2003). Recently, it was shown to have strong functional implications by increasing the robustness of oscillations in Cdk activity during the cell cycle (Pomerening et al., 2005). Yet, positive feedback does not make a system necessarily bistable. The emergence of this property depends on the detailed architecture of the regulatory network and is also very much influenced by the values of its component parameters, such as the sensitivity of its feedback loops. In order to connect the architecture of a regulatory module to its systems-level behavior, it is therefore crucial to measure the individual response of its subparts in a quantitative manner. In the present issue of Molecular Cell, Trunnell et al. (2011) use Xenopus egg extracts to quantify the response of Cdc25C hyperphosphorylation (which is required for its activation) to variable cyclin B1-Cdk1 activity, in order to evaluate the sensitivity of the Cdc25C feedback loop. To isolate this reaction from the rest of the cell-cycle machinery, the authors use a recombinant mutant of Cdk1 (Cdk1AF) that cannot be inactivated by Wee1 and does not need to be activated by Cdc25C but is still able to phosphorylate Cdc25C. By varying cyclin B1-Cdk1 activity with added nondegradable cyclin B1 (D65-cyclin B1), they show that the Cdc25C hyperphosphorylation is a highly ultrasensitive function of Cdk1 activity (i.e., it displays a sharp sigmoidal behavior), as revealed by the
Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc. 243
Molecular Cell
Previews
exceptionally high value The model also provides A Cdc25 (around 11) measured for a useful framework in which cyclin B1 the Hill coefficient. In addito test the functional imUltrasensitivity tion, the dephosphorylation portance of specific features Activation step of Ser 287 in Cdc25C of this regulatory module: cyclin B1 Cdk1 Cdk1 Cdk1 Mitotic entry cyclin B1 (which is also involved in for instance, it clearly shows Inactivation Cdc25C activation) displays that high ultrasensitivity in P Ultrasensitivity an even higher Hill coefficient Cdc25C phosphorylation of about 32. strongly contributes to make Wee1 Interestingly, Trunnell and the Cdk activation system B colleagues find that the ultrabistable (see gray plots on sensitivity in Cdc25C hyperFigure 1B), Most importantly, 100 phosphorylation can only be it reveals that, while only one partially recapitulated with a of the two positive feedback 80 more simple in vitro system. loops is enough to induce This reveals that, similarly as bistability, the presence of 60 in the Wee1 regulation loop both regulatory elements pro(Kim and Ferrell, 2007), vides a larger robustness of extrinsic factors in the exthis property to parameter 40 tracts—that yet remain to be modifications, and therefore n=24 n=11 determined—are likely to may buffer against evolutionn=6 20 n=3 n=1 enhance the nonlinearity in driven changes. the Cdc25C phosphorylation In summary, this study thus 0 process. Based on further further contributes to bridge 0 20 40 60 80 100 observations showing that the gap between the molecTotal cyclin B1 (nM) this nonlinearity can be comular properties of Cdk activapletely suppressed by mutation and the systems-level Figure 1. Ultrasensitivity and Bistable Response in the Activation of Cyclin B-Cdk tion of three out of the consequences on this canon(A) Sketch of the regulation of the activation of the cyclin B-Cdk complex, numerous known phosphorical cell-cycle transition. which involves reciprocal positive (Cdc25C) and double-negative (Wee1) ylations sites in Cdc25C, the feedback loops. authors conclude that the (B) Role of Cdc25 ultrasensitivity in promoting bistable transitions. The steadyREFERENCES state concentrations of active cyclin B1/Cdk1 as a function of cyclin B1 are observed in vitro ultrasensiplotted according to the kinetic model described in Trunnell et al. (2011). tivity arises from multisite Kim, S.Y., and Ferrell, J.E., Jr. (2007). Each gray plot corresponds to the steady-state solution for a given value of Cell 128, 1133–1145. phosphorylation, and three the Cdc25C Hill coefficient, as indicated on the graph. The red curve (nH = 11) and corresponding arrows display the low-to-high and high-to-low transidifferent plausible mechaNovak, B., and Tyson, J.J. (1993). tions in cyclin B-CDK activity as cyclin B is varied (hysteresis). nisms are proposed to J. Cell Sci. 106, 1153–1168. account for this observation. Pomerening, J.R., Sontag, E.D., and What may be the implications of such a very good quantitative agreement with Ferrell, J.E., Jr. (2003). Nat. Cell Biol. 5, 346–351. ultrasensitivity on the function of the bi- the previously reported hysteretic stable mitotic trigger? To answer this behavior for the steady-state activity of Pomerening, J.R., Kim, S.Y., and Ferrell, J.E., Jr. (2005). Cell 122, 565–578. question, Trunnell et al. turn to a kinetic cyclin B1-Cdk as a function of cyclin B1 model of activation of cyclin B-Cdk that level (Pomerening et al., 2003). It therefore Sha, W., Moore, J., Chen, K., Lassaletta, A.D., Yi, integrates the respective contributions of strongly suggests that cyclin B1-Cdk C.S., Tyson, J.J., and Sible, J.C. (2003). Proc. Natl. Acad. Sci. USA 100, 975–980. the Wee1 and Cdc25 feedback loops. switch-like activation upon mitotic entry This simple mathematical analysis, al- (and inactivation upon mitotic exit) are Thron, C.D. (1996). Biophys. Chem. 57, 239–251. most entirely constrained by experimen- such as to follow a complete hysteresis Trunnell, N.B., Poon, A.C., Kim, S.Y., and Ferrell, J.E., Jr. (2011). Mol. Cell 43, this issue, 263–274. tally determined parameters, displays cycle, as shown in Figure 1B (red curve). Steady-state active cyclin B1/Cdk1 (nM)
*
244 Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc.