Cell Division: Flipping the Mitotic Switches

Current Biology

Dispatches Cell Division: Flipping the Mitotic Switches Wolfgang Zachariae1,* and John J. Tyson2,* 1Max-Planck-Institute

of Biochemistry, Martinsried, Germany of Biological Sciences, Virginia Tech, Blacksburg, VA, USA *Correspondence: [email protected] (W.Z.), [email protected] (J.J.T.) http://dx.doi.org/10.1016/j.cub.2016.11.007 2Department

Alternation between DNA replication in the mother cell (S phase) and equal partitioning of the replicated chromosomes to the daughter cells (M phase) during eukaryotic cell division is governed by switches that flip protein kinases on and off. New work reveals that the M-phase promoting kinase is opposed by a phosphatase that also participates in a bistable switching mechanism.

To say that we understand the molecular basis of any aspect of cell biology is to provide a clear and convincing molecular explanation of the characteristic physiological features of the phenomenon. For example, the cell division cycle in eukaryotes proceeds through a fixed, circular sequence of four phases: G1 (unreplicated chromosomes) to S (DNA synthesis) to G2 (replicated chromosomes) to M (mitosis) and back to G1. Under ordinary circumstances, the transitions from one phase to the next are ‘irreversible’, e.g. the cell does not slip back to do a second round of DNA replication before dividing or try to carry out two mitotic divisions without an intervening S phase. It is now recognized that the irreversibility of the G2-to-M transition is due to ‘bistability’ in the molecular mechanism that activates the M-phase promoting kinase. A new study published in this issue of Current Biology by Mochida et al. [1] shows that the G2-to-M transition is actually governed by a pair of bistable switches: one controlling the kinase and the other controlling its counteracting phosphatase. During the triumphant decades of molecular cell biology (1980–2000), it was discovered that the progression of eukaryotic cells through the DNA replication/division cycle is governed by the orderly phosphorylation and dephosphorylation of a large number of proteins (the ‘substrates’ in Figure 1) by a particular class of ‘cyclin-dependent’ protein kinases [2,3]. The first of these kinases, originally called MPF, for M-phase promoting factor, was confirmed to be a heterodimer of Cdk1 (the catalytic subunit) and cyclin B

(a regulatory subunit). Wherever we look among Eukarya, we find this kinase complex playing a key role as cells enter mitosis. To exit mitosis, the kinase activity must be killed (usually by degradation of the cyclin component), and the phospho-groups must be removed from the mitotic substrates by counteracting protein phosphatases. It was recognized early on that, while cyclin B levels increase steadily during S and G2 phases, the kinase activity of the Cdk1–CycB complex rises sharply as cells enter M phase. Yeast geneticists, led into battle by Sir Paul Nurse [3], discovered a regulatory network controlling the activity of MPF. A kinase called Wee1 inhibits MPF by phosphorylating Cdk1 on Tyr15, while a counteracting phosphatase called Cdc25 activates MPF by removing this inhibitory phosphorylation. Surprisingly, it was quickly discovered that Cdk1 regulates its own regulators by inhibiting Wee1 and activating Cdc25 through phosphorylation (Figure 1). That this seemingly simple three-enzyme system is capable of generating complex regulatory output of relevance to cell-cycle progression was only realized when Novak and Tyson studied a mathematical model of this network (highlighted as 1 in Figure 1 [4]. Their analysis revealed that the Wee1–Cdk1–Cdc25 system constitutes a bistable switch, which explains both the abrupt activation of Cdk1 required for M-phase entry and the irreversibility and directionality of cell-cycle progression into and out of M phase. Confirming this model required

R1272 Current Biology 26, R1272–R1296, December 19, 2016 ª 2016 Elsevier Ltd.

careful measurements of concentrations and enzyme activities as well as methods to manipulate them, and thus took many years [5,6]. Once confirmed, however, the concept of bistability changed our view of cell-cycle control from smooth oscillations of Cdk1 activity to a ratchet-like sequence of low and high Cdk1 states [7]. (Sotto voce: this realization has yet to work its way into standard textbooks of molecular cell biology.) In hindsight, however, problems were lurking in this ‘kinase-centric’ view of cell-cycle control. At a time when cell-cycle research was dominated by analysing kinases, Cdk1-counteracting phosphatases were unknown and were therefore modelled as a constitutive enzymatic activity. This raises another problem: if the constitutive phosphatase is weakly active, then how can mitotic substrates be rapidly dephosphorylated as the cell exits mitosis and returns, in a matter of minutes, to G1 phase? If the phosphatase is strong throughout M phase, then the cell will be doing a lot of futile cycling of ATP when both the mitotic kinases and phosphatases are active. What are these mysterious Cdk1counteracting phosphatases and how are they regulated? One early candidate was Cdc14, an essential phosphatase in yeast cells, which is activated by an elaborate mechanism at the metaphaseto-anaphase transition [8]. But Cdc14 is not an essential gene in other organisms, and it does not seem to be the chief Cdk1-counteracting phosphatase in mitosis. That role belongs to protein phosphatase 2A and its regulatory subunit B55. PP2A–B55 is inhibited

Current Biology

Dispatches stoichiometrically by the phosphorylated form of alpha-endosulfine (ENSA-P), i.e., ENSA-P binds tightly to PP2A–B55 but is only slowly dephosphorylated (Figure 1) [9]. Hence, ENSA-P blocks PP2A–B55 from doing its job on other phosphorylated mitotic substrates. ENSA is phosphorylated by Greatwall (Gwl) kinase, which in turn is phosphorylated and activated by Cdk1– CycB [10,11]. By this pathway, Cdk1– CycB shuts down its counteracting phosphatase and prevents futile cycling during the first half of mitosis. When cyclin B is degraded as the cell exits mitosis, then PP2A–B55 activity could rise abruptly, but this would require that Gwl-P be dephosphorylated and inactivated. This problem can be solved if Gwl-P is also a substrate of PP2A–B55, creating a double-negative feedback loop, whereby Gwl-P inhibits PP2A–B55 (via ENSA-P) and PP2A–B55 inhibits Gwl-P by removing the phosphate group. From Novak and Tyson’s analysis of the Wee1–Cdk1–Cdc25 system, it is apparent that such a double-negative feedback loop can create a bistable switch (highlighted as 2 in Figure 1). The new work from Mochida, Novak and their collaborators [1] presents evidence for a second bistable switch governing M phase, which plays both a sufficient and necessary role in the irreversibility of entry into mitosis. Mochida et al. [1] started by inhibiting Wee1, which removes bistability from Cdk1 regulation (i.e., removes bistable switch 1), so that Cdk1 activity rises in direct proportion to cyclin B levels. Surprisingly, the phosphorylation of Cdk1 substrates (endogenous as well as luminescent probes) is still switch-like. By elegant biochemical reconstitution of the Gwl– ENSA–PP2A pathway from purified components and careful mathematical modelling of the molecular reaction system, the authors prove that subsystem 2 by itself constitutes a bistable switch (Figure 2). Thus, the phosphorylation of Cdk1 substrates and thereby entry into and exit from M phase is controlled by two bistable switches. The switches are intimately intertwined (Figure 1): switch 1 flips switch 2 to the phosphatase-OFF position by the Cdk1– Gwl–ENSA pathway, and switch 2 flips switch 1 to the kinase-OFF position by

B55

Wee1

PP2A

Cdc25

P

CycB

Cdk1

P Cdc25

Wee1

CycB

Cdk1

Gwl

1

Substrate 2

Gwl

ENSA

P

Substrate

P

P

ENSA

P

Tight reversible binding

B55

B55

PP2A

PP2A ENSA

P

Slow hydrolysis Current Biology

Figure 1. The two bistable switches (1 and 2) governing entry into and exit from mitosis. Cells enter mitosis when a variety of mitotic substrates (yellow) are phosphorylated by Cdk1–CycB and exit mitosis when these substrates are dephosphorylated by PP2A–B55. The activities of the ‘master’ kinase and phosphatase are governed by complex networks of subsidiary kinases and phosphatases. Solid arrows are chemical reactions, dashed arrows indicate the enzymes that catalyse each reaction. Opposing phosphorylation and dephosphorylation reactions are irreversible and involve futile cycling of ATP to ADP.

PP2A-mediated dephosphorylation of Wee1 and Cdc25. What are the implications of this discovery? Cdk1–CycB drives a cell into mitosis by phosphorylating substrates including regulators of the cytoskeleton, chromatin condensation factors, and components of the nuclear envelope. Bistable switch 1 results in a rapid and irreversible transition from G2 into M phase by activating Cdk1 and abruptly phosphorylating mitotic substrates. However, inactivation of kinase activity, by cyclin B degradation during anaphase, does not result in rapid substrate dephosphorylation unless the counteracting phosphatase is very active or, indeed, becomes very active when Cdk1–CycB activity

drops. We now know that the latter possibility is indeed the case. That high-kinase and high-phosphatase states of the control system are mutually exclusive also neatly avoids the problem of futile ATP hydrolysis during mitosis. But is bistability of the Gwl–ENSA– PP2A system required for robust mitotic entry or exit? Bistability offers stable (i.e., noise-resistant) ON and OFF states together with sharp, irreversible transitions between them (Figure 2). At the same time, bistability offers the possibility for regulators (external to the switching mechanism) to flip the switch. Activities that toggle the switch between its ON and OFF states, typically by unleashing a positive feedback loop,

Current Biology 26, R1272–R1296, December 19, 2016 R1273

Current Biology

Dispatches Kinase inhibitor

Low High Probe phosphorylation Low

Down

High Phosphatase activity

Low

High

Up Low

Kinase activity

High Current Biology

Figure 2. Bistability of the Gwl–ENSA–PP2A pathway. The ‘input’ activities of Cdk1 and Gwl kinases are controlled by a pharmacological inhibitor (staurosporine), and the ‘output’ activity of PP2A phosphatase is monitored by phosphorylation of a luminescent probe. If the control system is started in the low-kinase, high-phosphatase steady state and kinase activity is steadily increased (black rectangles), the switch flips to the low-phosphatase state at the Up threshold. Conversely, if the system is started in the low-phosphatase state and staurosporine is steadily added (red circles), then the switch flips at the Down threshold. In between these two thresholds, the control system is bistable. (This figure is a schematic representation of the model calculations and experimental data points in Mochida et al. [1].)

are called ‘starter’ activities. Mochida et al. [1] suggest that PP1 is a starter phosphatase for switch 2. While PP1 is known to be required for mitotic exit, it is PP2A that is thought to do the hard work of dephosphorylating most Cdk1 substrates. So, what is the role of PP1? PP1 is a phosphatase inhibited by Cdk1 phosphorylation, which PP1 itself removes. Thus, PP1 becomes active as Cdk1 activity drops, and PP1 then dephosphorylates Gwl [12]. Consequently, as PP2A rids itself of ENSA-P, the unphosphorylated form of ENSA is no longer quickly re-phosphorylated by Gwl. This effect allows PP2A activity to rise exponentially as Cdk1 activity drops, and for the cell to rapidly dephosphorylate mitotic substrates and enter G1 phase. Does switch 1 require a starter kinase? Originally, Novak and Tyson [4]

thought not; if the phosphatase opposing Cdk1-mediated phosphorylation of Wee1 and Cdc25 is not too strong, then the residual kinase activity of tyrosine-phosphorylated Cdk1 could be enough to ignite the positive feedback loops controlling switch 1. But the current results of Mochida et al. [1] make this scenario less likely, since PP2A–B55 is expected to be a powerful phosphatase in late G2 phase. Cdk2–CycA may play the role of a starter kinase for entry into M phase [13], but this is an issue that needs to be settled by further experimentation. Why two bistable switches? Mochida et al. [1] show that, in the absence of Wee1, phosphorylation of target proteins is still switch-like, due to bistability of the Gwl–ENSA–PP2A module. However, Cdk1 activation shifts to lower levels of cyclin B. In an egg, in which cyclin B is continuously synthesized, this shift dramatically advances entry into mitosis, threatening or even eliminating the separation of M phase from S phase; similar problems may also be faced by somatic cells. Thus, the Wee1–Cdk1–Cdc25 switch seems to be essential for proper regulation of the G2/M transition. Conversely, in the absence of the Gwl–ENSA–PP2A switch (i.e. in ENSA-depleted cells), substrate phosphorylation is slow and shallow. In cells, this sluggish phosphorylation of mitotic substrates would translate to a huge delay in entry into M phase, and this entry would likely be a mess because substrate phosphorylation reaches only half of normal levels. So, the Gwl–ENSA–PP2A switch appears to be essential for normal entry into M phase. The paper by Mochida et al. [1] provides an elegant example of the power of combining molecular biology with biochemical reconstitution experiments and mathematical modelling to reveal the complex behaviour and biological significance of molecular regulatory networks in eukaryotic cells.

R1274 Current Biology 26, R1272–R1296, December 19, 2016

REFERENCES 1. Mochida, S., Rata, S., Hino, H., Nagai, T., and Nova´k, B. (2016). Two bistable switches govern M phase entry. Curr. Biol. 26, 3361– 3367. 2. Murray, A.W., and Kirschner, M.W. (1989). Dominoes and clocks: the union of two views of the cell cycle. Science 246, 614–621. 3. Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344, 503–508. 4. Novak, B., and Tyson, J.J. (1993). Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos. J. Cell Sci. 106, 1153–1168. 5. Sha, W., Moore, J., Chen, K., Lassaletta, A.D., Yi, C.S., Tyson, J.J., and Sible, J.C. (2003). Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts. Proc. Natl. Acad. Sci. USA 100, 975–980. 6. Pomerening, J.R., Sontag, E.D., and Ferrell, J.E., Jr. (2003). Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2. Nat. Cell Biol. 5, 346–351. 7. Novak, B., Tyson, J.J., Gyorffy, B., and Csikasz-Nagy, A. (2007). Irreversible cell-cycle transitions are due to systems-level feedback. Nat. Cell Biol. 9, 724–728. 8. Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M., and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol. Cell 2, 709–718. 9. Williams, B.C., Filter, J.J., Blake-Hodek, K.A., Wadzinski, B.E., Fuda, N.J., Shalloway, D., and Goldberg, M.L. (2014). Greatwallphosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers. eLife 3, e01695. 10. Mochida, S., Maslen, S.L., Skehel, M., and Hunt, T. (2010). Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science 330, 1670–1673. 11. Gharbi-Ayachi, A., Labbe, J.C., Burgess, A., Vigneron, S., Strub, J.M., Brioudes, E., VanDorsselaer, A., Castro, A., and Lorca, T. (2010). The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A. Science 330, 1673–1677. 12. Heim, A., Konietzny, A., and Mayer, T.U. (2015). Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit. EMBO Rep. 16, 1501–1510. 13. Guadagno, T.M., and Newport, J.W. (1996). Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity. Cell 84, 73–82.