The Cell Cycle

[email protected] http://www.gurdon.cam.ac.uk/~pineslab/ New_Web_Site/Site/Lectures.html What does a cell need to do to proliferate?

Chromosome Separation

Product - Substrate?

Oscillator?

DNA replication How do we know there is a cell cycle? G1 or S or G2 M M

+ =

G1 S S + =

S G2 G2 delay + =

Rao & Johnson, 1970, Nature 225, 159-164; Johnson & Rao, 1970, Nature 226, 717-722 After Murray and Hunt, 1993 M phase and S phase are different cellular states Problems to be solved: Alternation and Completion Chromosome Separation

M Murray and Hunt, 1993 The Cell Cycle: an introduction.

Morgan, D.O., 2007 The Cell Cycle: Principles of Control S

DNA replication Cyclin-CDK complexes drive the cell cycle

• Three converging lines of evidence: Yeast genetics Xenopus meiosis Translational control in sea urchin eggs

‘M phase’ ‘S phase’ Cyclin-CDK Cyclin-CDK

S How can you isolate cell cycle regulators?

Schizosaccharomyces pombe (ﬁssion yeast): Position in cell cycle related to length of cell

Screen for genes that accelerate or slow down the cell cycle cdc2 is a conserved protein kinase required at 2 points in cell cycle and with a wee allele

Wee1, mik1 cdc25

cdc2 cdc13 (Cyclin B)

Interphase Mitosis

After Murray and Hunt, 1993. MPF: a potent trigger for mitosis Xenopus laevis: arrested in G2 of meiosis I, egg arrested in metaphase of meiosis II

Factor in egg cytoplasm forces oocyte to enter M phase - M phase promoting factor (MPF)

Self ampliﬁes and does not require protein synthesis: pre-MPF in oocyte

Universal property of M phase cells

Progesterone Inject cytoplasm

Masui and Markert, 1971, J. Exp. Zool 177, 129. After Murray and Hunt, 1993. Cell 390

Unfertilized Fertilized A23187 NH&I UF abcdefghi jk abcdefghi jk abcdefghiik abcdefghij kkk Cyclins: Coincidence or cause? -xSea urchins: large eggs arrested in G1 of ﬁrst mitotic cell cycle 116 Fertilisation causes large increase in translation One protein only translated after fertilisation and destroyed at each mitosis: Cyclin 68

Cyclin

-Y 35 MPF S M S M -2 Evans et al. 1993. Cell 33, 389 MPF eventually shown to consist of cyclin and Cdc2 (Dorée and Hunt, 2002, J Cell Sci, 115, 2461-4)

Figure 1. The Patterns of Protein Synthesis in Eggs before and after Activation ?S-methionrne was added at a final concentration of 30 &i/ml to 15 ml of a suspension of 20,000 unfertilized eggs per milliliter. After 5 min, portrons of this suspension were fertilized, or made 10 pM in A23187 by addrtion of IO mM stock in drmethylsulfoxide. or made 10 mM in NH, Cl by addition of 1 M stock. Samples were taken for analysrs at 10 mm intervals starting at 25 min (lanes a) until 115 min (lanes j) after addition of the activator. Samples (lanes k) were taken at 127 min. The last two lanes labeled Uk and Fk are lighter exposures of the last unfedilrzed and fertilized lanes, respectively. The exposure of the autoradiograph in the unfertilrzed panel was four times longer than the other three to compensate for the low level of incorporation before fertilization. Bands X, Y, and Z are marked as examples of polypeptides whose synthesis IS reduced after fertrlization; and A, B. and C as the major nonhrstone proterns whose synthesis is activated. ing change is the appearance of the prominent new bands however, all the proteins are less strongly synthesized than A, B, and C after fertilization or activation with A23187, in activations that involve release of intracellular calcium. much as happens in Spisula (Rosenthal et al., 1980). The Synthesis of Some Proteins Stops after However, closer inspection reveals other interesting fea- Fertilization tures, of which the most unexpected is the behavior of Several proteins, like the examples labeled X, Y, and Z on protein A, which we shall call “cyclin” henceforth. It is the Figure 1, are made in the unfertilized egg but are very hard most strongly labeled protein at early times after fertiliza- to detect after fertilization, though they continue to be tion, but by 85 min after fertilization (lane g, fertilized) it made in ammonia-activated eggs. These comparisons are has almost disappeared. It gets stronger again in lanes h most easily made by examining the last three lanes of and i, only to decline again in lane k. These oscillations in Figure 1, in which light exposures of the final lanes (k) from the level of cyclin are extremely reproducible, as can be unfertilized (U) and fertilized (F) eggs are matched with the seen in Figures 2, 3, and 6, which show similar behavior final ammonia-activated lane. The decline in synthesis in in different batches of fertilized Arbacia eggs. prefertilization bands is easier to see in Figure 7, which shows a similar experiment in Lytechinus pictus. Cyclin Does Not Oscillate after Parthenogenetic Activation Cyclin Is Destroyed Periodically at a Particular Although cyclin synthesis is strongly induced by A23187 Point in the Cell Cycle or NH&I treatment of unfertilized eggs, under neither of Figure 2 correlates the oscillations in the level of cyclin with these circumstances does its level oscillate in the same the cleavage cycle. The experiment was conducted in the way as in fertilized eggs. This is probably because the same way as the previous one, except that additional eggs do not divide, a point enlarged upon below. samples from the suspension of fertilized eggs were fixed in 1% glutaraldehyde for later examination under the mi- Ammonia Activation Does Not Turn On the croscope. The dashed line in Figure 2 denotes the cleav- Synthesis of All the Proteins age index, measured as described in Experimental Pro- As mentioned above, activation of eggs with weak bases cedures. The other two curves show the relative intensities like NH4+ causes only about half the stimulation of protein of cyclin and protein B, as determined by densitometry of synthesis that A231 87 or fertilization gives. Is this because the autoradiographs. Label accumulates more or less lin- the synthesis of all the proteins is half turned on, or early in protein B, whereas cyclin (band A) falls precipi- because half the proteins are fully turned on? Figure 1 tously at the onset of cleavage, only to rise and fall again shows that protein B did not appear after ammonia acti- during the second cell cycle. Label in protein C also rose vation, though it is one of the most strongly labeled in linearly, but the data have been omitted from this figure fertilized and ionophore-activated eggs. For the most part, for clarity of presentation. Cyclin-CDK complexes deﬁne the state of the cell cycle

Mitosis Initiation

cdc13-cdc2 M

Degrade cdc13 or cdc2

G2 G1 START

S Re-initiate DNA synthesis

DNA Synthesis Initiation DNA Synthesis cig2-cdc2

Hayles et al., 1994, Cell 78 , 813 and Broek et al., 1991, Nature 349 , 388. The cell cycle as alternation of CDK activity

Low CDK activity: Assemble and ﬁre origins of replication

High CDK activity: Repress origins of replication

Build mitotic apparatus

S. pombe paradigm - the Threshold hypothesis Stern and Nurse, 1996, Trends Genet. 12, p345-350

Norton and Difﬂey, 2000, Molecular Cell, 5, 85-95 Testing the Cdk threshold hypothesis

Cyclin B (Cdc13) Analogue Sensitive Cdc2 Kinase

Cyclin-cdc2 fusion drives cell cycle in absence other cyclins

Control amount of kinase activity through analogue sensitive Cdc2

Low concentrations of inhibitor block mitosis

High concentrations block DNA replication

Add different inhibitor concentrations to drive cell cycle with non-degradable Cdc13

Block G2 cells and release into low concentrations of inhibitor - Cells re-replicate

Coudreuse & Nurse, 2010 Nature 468, 1074-1079 The Cell Cycle: Alternation

Metaphase

Initiation of Mitosis Cyclin B1-CDK1 G0 Cyclin A-CDK1/2 M

G2 G1 START or R point Cyclin D-CDK4/6

Completion of DNA Synthesis S Initiation of DNA Synthesis DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK2 Waves of cyclin-CDK kinase activity during the human cell cycle

G1 S G2 M G1

Cyclin B-CDK1

Cyclin A-CDK2 Cyclin E-CDK2 Kinase activity

Cyclin D-CDK4/6

time M A

Growth Factors Cdc25 But Cdk2 is Not Essential for Mitosis

Ortega et al., 2003, Nature Genetics, 35, p25-31

Cyclins E and A are partially redundant

Cyclin E is only essential for endo-replication

Geng et al., 2003 Cell 114, 431-443

Cyclin A is only essential in early embryos & stem cells

Kalaszczynska et al., 2009 Cell 138, 352-365 Most mitotic cycles only require one Cdk

Santamaria et al., 2007, Nature 448, 811-815 • Looks like dividing animal cells are just like ﬁssion yeast • But for cells to differentiate and form particular tissues they need specialised Cdks • Cdk2 - spermatogenesis, oogenesis • Cdk4 - pancreatic beta cells, pituitary, mammary epithelium • Cdk4 or Cdk6 - haematopoesis, cell size Why are there multiple Cyclin-Cdks?

• Partial Redundancy? S. cerevisiae paradigm

• Tissue speciﬁc roles The Cell Cycle: Alternation

Metaphase

Initiation of Mitosis Cyclin B1-CDK1 G0 Cyclin A-CDK1/2 PoloM Aurora G2 G1 Cyclin D-CDK4/6

Completion of DNA Synthesis S Initiation of DNA Synthesis START or R point DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK1 & 2 Coordination between mitotic kinases

Kinase Domain PBD PBD

• Polo box binds to S-pT or S-pS - Elia et al., 2003, Science 299, p1228 • Often generated by Cdk (S-T-P) • Thus Cdk phosphorylation generates Plk substrate

Eg: Cdk1 phosphorylates INCENP to recruit Plk1 to kinetochores

- Goto et al., 2006, Nature Cell Biology 8, p180 Regulating a CDK

Cyclin Proteolysis (Ubiquitin) Cyclin Binding

Thr14 & Tyr15 Phosphorylation (wee1/mik1) Thr14 & Tyr15 dephosphorylation (Cdc25)

T-loop Thr dephosphorylation (p24KAP) T-loop Thr phosphorylation (CAK)

Inhibitor binding Inhibitor removal

T14 Y15

Deactivate Activate CDK

T160 Morgan 1995, Nature 374, 131. Cyclin-CDK inactivation: the paradigm

N-term

P P ATP Cyclin P Box KINASE Cyclin Fold 2

C-term

Cks OONFF Wee1 - Generating a Robust Switch

• Fission yeast wee1 and mik1 prevent premature mitosis • mik1 stabilised by unreplicated DNA

• Wee1 in animal cells blocks mitosis in interphase

Ultrasensitive response to inhibition by Cdk1 - bistable state

SP SP SP SP SP Kinase Domain Sites not Conserved well conserved inhibitory sites Not inhibitory Kim & Ferrel, 2007, Cell 128, 1133-1145 Cyclin-CDK inactivation: the Paradigm 2

Cki

N-term

ATP Cyclin P Box KINASE Cyclin Fold 2

C-term

Cks OONFF CKIs inhibit both the CDK and the cyclin

Russo et al, 1996 Nature 382, 325. Yeast CKI Paradigm: Cell Cycle Co-ordination - Sic1

Cln2-Cdc28 Clb5-Cdc28

Cdc4 Cdc34 - SCF Sic1 Sic1 p40 p40 Degradation

Cdc28-Clb2 Cdc28-Clb5/6

M G1 S Phase RESEARCH LETTER in the assay (Supplementary Fig. 2f) and in mathematical simulations S76 and S80), and this specificity depended on the RXL2 and RXL3 (Supplementary Fig. 3). This processive pattern argues against the cur- docking sites in Sic1 (Fig. 2b; note that in all figure labels, the indicated rent model of ultrasensitivity in the Sic1 phosphorylation switch, which Cdk sites are those left unmutated, unless otherwise indicated). Cln2– is based on the assumption of a distributive mechanism with equal Cdk1, on the other hand, showed a preference for the N-terminally specificity of different sites1,15. located site T5 (Fig. 2b). Thus, docking interactions direct the asso- To dissect the mechanism of the processive multiphosphorylation ciated kinase to a small number of primary phosphorylation sites. We cascade, we first studied the impact of potential docking interactions speculate that these primary sites interact with Cks1 to drive processive between Sic1 and cyclins. In previous studies, we found that rapid Sic1 phosphorylation of additional sites. phosphorylation by Clb5–Cdk1 depends on an interaction between With these primary specificities in mind, we set out to map the RXL motifs in Sic1 and the hydrophobic patch docking site (hp) in pathways along which Cln2–Cdk1 and Clb5–Cdk1 catalyse the phos- Clb5; a triple mutation in this site (Clb5hpm) decreases the net phosphorylation of the critical sites required for Sic1 degradation. The phorylation rate (Supplementary Fig. 4a–h)16. Further, we found here original model of Sic1 regulation proposed that six or more sites must that a version of Sic1DC with mutations at its four RXL motifs (Sic1DC- be simultaneously phosphorylated in vivo to facilitate binding of 1234rxl) showed less abrupt production of multiphosphorylated species phospho-Sic1 to the SCF subunit Cdc4 (ref. 1). On the other hand, by Clb5–Cdk1, showing that processive multiphosphorylation requires later binding studies revealed that closely positioned pairs of phos- both Cks1-dependent and hp-dependent docking (Supplementary Fig. phorylation sites (pT5/pS9, pT45/pT48, or pS76/pS80; see Fig. 2a) 4i). Cln2–Cdk1 had only a mild RXL effect on the phosphorylation each present separate entities with a strong affinity for Cdc4, indicating pattern, probably because Cln2 does not contain a conventional hp like that just two phosphorylation sites, in the right positions, might be that in the B-type cyclins. In recent studies, we also located a ten-amino- sufficient for Sic1 degradation17. Our results provided a way to recon- acid stretch in Sic1, 136VLLPPSRPTS145, which confers Cln2 specifi- cile these findings: we proposed that the requirement for six or more city16. Here we found that a five-alanine mutation of the first five sites in vivo reflects a requirement for priming phosphorylation events hydrophobic residues in this stretch, or a synthetic competitor peptide that direct processive phosphorylation of critical phosphodegrons. To containing the docking site, reduced the abrupt multiphosphorylation test this possibility, we first measured phosphorylation of a Sic1DC pattern for Cln2 (SupplementarySic1 Fig. 4j). Inactivation In conclusion, both Clb5– hasmutant Parallels with all Cdk with sites changed Wee1 to alanine except for the triple Cdk1 and Cln2–Cdk1 use docking mechanisms, in addition to Cks1, to cluster S69/S76/S80, which contains two potential paired degrons achieve processive multiphosphorylation of Sic1. Inactivation(S69/S76 and S76/S80). There was no processive multiphosphoryla- Using Sic1DC mutants carrying only one Cdk site (Fig. 2a, b), we tion of the cluster S69/S76/S80 (Fig. 2c, lane 2 in each panel), but found that Clb5–Cdk1 rapidly phosphorylated just four sites (T5, T33, processivity could be induced by adding back single Cdk1 sites to

a MTSPFNGLTSPQRSPFPK Sic1ΔC stop at 215 T2 S9 T45 S69 S80 VLLPP

T5 RXL1 T33 T48 S76 RXL2 RXL3 RXL4 T173 S191 Inhibition domain

MTPSTPPRSRG PVTPSTTKSFK

Cln2–Cdk1 Clb5–Cdk1 T2 T5 T33 T45 S69 S76 S80 T173 S191 H1 b c Sic1 C-S69/S76/S80 cluster Sic1 C-S69/S76/S80 cluster Sic1ΔC Δ Sic1ΔC Δ Sic1ΔC WT – T5 T33 T45T2/T5 WT – T5 T33 T45T2/T5 Sic1ΔC -234rxl 6P-... 5–6P Sic1ΔC -134rxl Sic1 is processively phosphorylatedClb5 4–5P by Cln2 and Clb5 3–4P Sic1ΔC -124rxl 1–2P Sic1ΔC -123rxl Clb5 phosphorylation generates1P a positive feedback loop

Sic1ΔC Cln2

d e g T2/T5/T33/T45/S69/S76 Sic1mutant Gal Glc Cln2 Clb5 T2/T5/T33/T45/S76 /S80 Wild type T33/ T33/ 2P– T45/ T33/ T45/ T33/ nP S69/S76/S80 T48A T45 T48A T45 T5/S69/S76/S80 1P* T33/S69/S76/S80 T45/S69/S76/S80 0P Time 010203040506070 010203040506070 T33/T45/S69/S76/S80 (min) Kõivomägi et al., 2011, Nature 480, 128–131 Sic1mutant Gal Glc T2/T5/T33/T45/T48A/S69 f T2/T5/T33/T45/T48A/S76 /S76/S80 T33/T45/S69/S76/S80 T33/T45/S69/S76/S80-T48A 2P– T33/T45/S69/S76/S80-(47-49)AAA nP T33/S69/S76/S80 1P* T33/T45/S69/S76 0P T33/T45/S76/S80 Time (min) 010203040506070 010203040506070 Figure 2 | Phosphorylated priming sites provide docking interactions for of sites described in c were overexpressed under the galactose promoter to assay efficient phosphorylation of suboptimal sites in phosphodegrons. the ability of cells to degrade Sic1. Gal, galactose; Glc, glucose. e, Comparison of a, Schematic view of phosphorylation sites, docking motifs (Clb5- and Cln2- the in vitro phosphorylation profiles of Sic1DC versions containing only the specific), phosphodegrons (ovals17) in Sic1. b, Phosphorylation specificity of phosphorylation sites T33/T45 or T33/T45 with mutation T48A. f,The Clb5–Cdk1 and Cln2–Cdk1 towards different Cdk sites was studied using nonconsensus Cdk1 site T48 is important for viability of cells overexpressing Sic1DC constructs containing a single fixed Cdk site. For Clb5–Cdk1, the Sic1. The same assay as d was used. In panels d and f, the labels indicate dependence of the site-specificity profile on RXL docking sites was assessed unmutated amino acids, and all other consensus Cdk sites are mutated; using Sic1DC constructs containing a single Cdk site and a single fixed RXL mutations in the nonconsensus Cdk sites are highlighted in red. g,The motif. c, The impact of different priming phosphorylation sites on cooperative phosphorylation and degradation dynamics of Sic1 were followed after the phosphorylation of the degron cluster S69/S76/S80. Phospho-site mutants of release of cells from a-factor in a system constitutively expressing mutated Sic1DC carrying the intact S69/S76/S80 cluster and the indicated sites left versions of noninhibitory haemagglutinin-tagged Sic1DC–3HA. The asterisk unmutated were used in a kinase assay with Cln2–Cdk1 and Clb5–Cdk1 using indicates a G1-specific phosphorylation by an unknown kinase. Phos-tag SDS–PAGE. d, Full-length Sic1 versions containing the combination

ReviewGenerating a Robust Switch also requires Cell cycle control by protein phosphatases Regulating Antagonistic Phosphatasesreview

Figure 2. The Diversity and Complexity of PP2A Numerous mechanisms control PP2A. Left: PP2A exists predominantly as a heterotrimer, with conserved A and C Avoid futile cycles! subunits and variable B subunits. This heterotrimer is Sidebar A | In need of answers regulated at multiple levels, including regulation of hetero- (i) It will be crucial to identify the phosphatases that dephosphorylate trimer assembly, microbial toxins (such as okadaic acid and microcystin), protein inhibitors such as SET and CIP2A, and ADP Substrate + PO4 Gwl and Ensa/ARPP19 to explain how the Gwl–Ensa/ARPP-19 phosphorylation of the B and C subunits to regulate activity, pathway is switched off, or reset, for the next round of the cell cycle. assembly, and targeting. Right: the B subunits are encoded by at least 15 different genes, each with multiple splice (ii) It will be also important to explore the role of the Gwl–Ensa/ARPP-19 Either/Or system in various biological contexts—such as the mammalian Kinase Phosphatase variants. nervous system—and in various organisms (yeast and nematodes NOT both at once compared with insects and humans). (iii) More generally, as it is becoming clear that protein phosphatases can ATP Substrate + OH PO4 be highly and specifically regulated, we need to elucidate the details density (PSD) but show different patterns of phos- of their control mechanisms, especially in terms of the balance with phorylation. Most importantly, mutant mice lacking their partner kinases. Fig 1 | Ensuring productive cycles. A protein kinase adds, whereas a one or the other of these two PP1 regulators show (iv) How many other of the PPP family of phosphatases can be switched phosphatase removes, phosphate residues on substrates. If these mutually distinct phenotypes (Allen et al., 2006; Feng et al., on and off? Only biochemistry will tell! antagonisticHow enzymes to work turn simultaneously, off a specific it not only results complex? in a waste of 2000; Wu et al., 2008), strongly suggesting that (Egloff et al., 1997), but sequences flanking this motif also play PP1 complexes containing these proteins catalyze distinct ATP, but also renders impossible a full switch-like interconversion of the a key role in defining the affinity and specificity of these regula- dephosphorylation events to modulate synaptic plasticity. phosphorylation state of the substrate. To avoid this, the two enzymes should tors for PP1 isoforms (Carmody et al., 2004; Terry-Lorenzo Thus, subtle differences in the associated regulatory subunit functions of a particular phosphatase complex, it is necessary to iden- work alternatively, ideally while communicating with each other. et al., 2002). There are additional PP1-binding domains that can direct the phosphatase to different substrates or sites tify the appropriate regulatory subunit. Of course, these inhibitors can allow a single PP1 catalytic subunit to recruit more than one even in the same subcellular compartment. still provide useful clues if they are used carefully. regulator, and several heterotrimeric PP1 complexes have beenphosphatase observed that (Eto dephosphorylates et al., 2002; Lesage CDK et substrates. al., 2007). There These must be Is PP2A-B55δ the phosphatase for CDK substrates? dataother combined phosphatases with bioinformatic acting at mitotic analysis exit. of For all definedexample, PP1- a formCellular of PP2A Complexes Our attention was drawn to the question of which phosphatase(s) bindingPP1 is a domains good candidate, suggest thatas a theconsiderable full complement body of of data cellular has alreadyPP2A, like PP1, functions solely as a multimeric enzyme. The were responsible for mitotic exit by the discovery that PP2B/ complexes,implicated generatedit in mitotic by exit. combinatorial Cdc14 could assembly be another of PP1 candidate,PP2A catalytic subunit makes up to 0.1% of total cell protein calcineurin was activated when crude Xenopus egg extracts are catalyticalthough and the regulatoryfunctions of subunits, Cdc14 homologues may exceed in morehigher than eukaryotesand is encoded by two human genes (PPP2CA and -B). The released from CSF arrest (arrested at meiotic metaphase II with high 100remain enzymes unclear that [4,28,29]. control different aspects of mammalian cell PP2A catalytic (or C) subunits are usually associated with CDK activity by a combination of Mos kinase and Erp1/Emi2) by physiology. a scaffolding A-subunit and one of a large array of regulatory Recent structural studies have provided new insight into how B-subunits (Figure 2). Eukaryotic cells contain a large variety of the addition of CaCl2 [12,13]. Inhibition of PP2B (by cyclosporin Greatwall kinase regulates PP2A-B55δ activity in mitosis A) seriously delayed the return to interphase in this setting, and we phosphataseThe Greatwall holoenzymes (Gwl) gene recognize was originally substrates. identified The as cocrystal- the Scant (Scottthese PP2A complexes. PP2A is an essential enzyme, and designed a substrate that could monitor phosphatase activity in lizationof the of Antarctic) PP1 catalytic mutation subunit in withDrosophila the M110 [30]. or myosin Scant light- later turnedknockdown of the catalytic or a subset of regulatory subunit these crude extracts. To our surprise, we found that the main role chain-bindingout to encode subunit a protein (also kinase named that MYPT1 is important and PPP1R12A) for mitosis [31].genes results in apoptosis (Gotz et al., 1998; Kong et al., 2004; of PP2B/calcineurin was to allow the activation of a second phos- demonstratedDrosophila mutants that the juxtapositiondeficient in ofGwl the showed M110 anykrin defects repeat in chromoLi et- al., 2002; Silverstein et al., 2002; Strack et al., 2004). There domain to the PP1 catalytic center significantly extends the phatase, not calcium activated, that was normally highly active some condensation and delayed cell cycle progression throughoutare four B subunit gene families, each with two to five genes and substrate-binding site (Figure 1)(Egloff et al., 1997; Terrak many with multiple splice variants. A small fraction of PP2A also in interphase and inhibited in meiosis and mitosis (Fig 2A; [12]). late G2 phase to mitosis. The kinase activity of Gwl increases as cells et al., 2004). The substrate recognition platform formed by the exists as heterodimers, with the catalytic subunit bound to other This regulation of phosphatase activity was abolished after addi- enter mitosis, during which Gwl itself is highly phosphorylated, at binding of diverse regulators with the catalytic subunit provides proteins such as the a4 subunit. A recent high-density mass tion of buffer to the concentrated egg extracts, so we had to use anleast attractive in part explanation by CDK1. forFurther the differing analysis specificity using Xenopus of phospha- egg extractsspectrometry analysis confirms that additional proteins can an immuno depletion technique—instead of standard biochemi- taserevealed multimers. that Gwl A similar is not model only important for recruiting for substrates entering mitosis, applies butreplace also B subunits, and still others interact tightly with known cal fractionation and/or purification methods—to identify the torequired PP2A asfor wellmaintaining (Figure the 2). CSF-arrested The M110:PP1C mitotic complex state [32]. has If Gwlheterotrimers is (Goudreault et al., 2009). The diversity of PP2A fluctuating phosphatase activity. It proved to be a particular form additionaldepleted mechanismsfrom mitotic to egg regulate extracts its (CSF), activity. active For example,CDK1 is inactivated the derives from the fact that cells can assemble over 200 biochemi- of PP2A that contained a B55δ regulatory subunit [15]. In extracts M110:PP1Cby inhibitory complex phosphorylation binds and regulates on its Tyr the 15 polo-like residue, kinase-1 rather thancally by distinct complexes containing different combinations of A, that had been depleted of PP2A-B55δ, mitotic phosphorylation was (PLK1)cyclin (Yamashiro proteolysis et [32]. al., 2008 These). The findings ability suggest of M110:PP1C that the to role bind of GwlB, C,in and other subunits. accelerated at a lower-than-usual concentration of cyclin B [15]. PLK1,mitosis however, is to control requires the CDK1 the mitotic regulators phosphorylation Cdc25 and/or of M110Wee1, whichTo optimize this diversity, organisms express different regula- Furthermore, histone 1 kinase activity, which reflects the level of (calledare themselves MYPT1 in substrates this study) of by CDK1 CDK1. [33–35]. These studies Strikingly, highlight even intory the subunits in different tissues at different times, leading to the Cdc2 kinase, was enhanced when PP2A-B55δ was depleted in thepresence potential of of high covalent CDK1 modifications activity, loss of Gwl regulatory induces subunits dephosphoryla to presence- of different PP2A complexes in different mammalian interphase egg extracts. This is reminiscent of the experiments lead- inducetion of the mitotic conformation phosphoproteins, changes that strongly modulate suggesting their interaction that Gwltissues acts (McCright et al., 1996; Moreno et al., 2000; Strack ing to the characterization of INH [25]. INH was originally defined withas an both inhibitor the phosphatase of the protein catalytic phosphatase(s) subunit (discussed that antagonize below) CDK1.et al., 1998). Moreover, as with PP1, the PP2A regulatory and the substrate. as an activity that inhibited the activation of MPF in Xenopus Indeed, PP2A-B55δ is activated after depletion of Gwl from subunitsCSF- provide for exquisite substrate specificity, directing It is noteworthy that many mammalian PPP regulators exist as oocytes, and was later identified as a form of PP2A [26,27]. We arrested mitotic extracts [36,37]. Oddly, however, Gwl doesthe not dephosphorylation of different phosphorylated residues on multiple gene products and these isoforms, while sharing many the same protein. For example, the PP2A heterotrimer contain- found that depletion of PP2A-B55δ led to a failure to dephospho- phosphorylate any subunit of this phosphatase complex, so its mode key biochemical properties, serve distinct physiological func- ing a B (PPP2R2) subunit dephosphorylates threonine-124 of rylate mitotic CDK substrates at the end of mitosis, although cyclin of action was unknown for some time. tions, further diversifying the functions of cellular PPP SV40 large T antigen, while a PP2A heterotrimer containing B00/ degradation and CDK inactivation took place more or less normally complexes. For example, the two isoforms of the neuronal PR72 (PPP2R3) dephosphorylates the immediately adjacent [15]. These observations initially suggested that PP2A-B55δ was the actin-bindingEnsa/ARPP-19, proteins, the neurabin-I first Gwl and substrates, neurabin-II inhibit (or spinophi- PP2A-B55serines-120δ and -123 in the same substrate (Cegielska et al., main phosphatase for CDK substrates. However, when B55δ was lin),Two both small, bind heat-stable PP1g1 and proteins are concentrated called Ensa at and the ARPP-19 postsynaptic have almost1994). depleted after the egg extracts entered mitosis, it was no longer 70% sequence identity and are members of a highly conserved pro- required to exit mitosis [15]. This puzzling result suggested that tein family (Fig 2B). ARPP-19 and its short form ARPP-16 were first although PP2A-B55δ is required for mitotic exit, its crucial role for identified as major substrates for protein kinase A in brain tissue Molecular Cell 33, March 13, 2009 ª2009 Elsevier Inc. 539 mitotic exit is already complete before entering mitosis. We have [38,39]. Therefore, they seemed to be involved in dopamine signal- no idea how PP2A-B55δ affects mitotic exit during the preced- ling in the postsynaptic neuron, where signalling cascades using pro- ing interphase, nor how many CDK substrates are dephosphoryl- tein phosphorylation are important [40]. Ensa was initially thought to ated by PP2A-B55δ. In any case, PP2A-B55δ is clearly not the only be an endogenous ligand for the sulphonylurea receptor and was

Fig. 3. Phosphorylation and function of Ensa in Xenopus egg extracts. (A)Proteins from Xenopus egg extracts arrested in the indicated cell-cycle stages were supplemented with or without His-tagged recombinant WT or S67A mutant Ensa. Recom- binant (added) or endogenous Ensa was detected by immunoblotting with antibodies to Ensa (top) or phospho-Ser67 (middle). The same set of samples was also analyzed by SDS–polyacrylamide gel electrophoresis in the presence of Phos-Tag (bottom). The migration positions of dephosphorylated [Ensa-OH, added-OH, or Gwl-phosphorylated (Ensa-PO4,added-PO4]proteinsareshown.(B)EndogenousEnsawasimmunode- pleted (lane 2) from fresh cycling extract and either WT (lane 3) or S67A mutant (lane 4) of recombinant Ensa added back. (C to E)Eggextractsshownin(B)were incubated at 23°C, and aliquots were takenat7-minintervalsforanalysis.Apc3 (upper panels), cyclin B2 (middle panels), and phosphorylation of Tyr15 of Cdc2 (lower panels) were detected by immunoblotting. Histone H1 kinase (black squares) and MBP-Fzy-Ser50 phosphatase activities (red circles) were measured and plotted (70 min of H1 kinase and 0 min of Ser50 phosphatase activities of mock extract taken as 100%). (C) Mock depletion; (D) Ensa-depleted; (E) Ensa-depleted with WT Ensa added

back. See fig. S3 for additional data fromthisexperiment.Arrows indicate the on December 16, 2010 mobilities of interphase and mitotic Apc3. A.U., arbitrary units.

Cell cycle control by protein phosphatases reviewGenerating a Robust Switch - Target a Speciﬁc Phosphatase Complex with an Inhibitor

alent activity that dephosphorylated Tyr15 was ac- tracts, consistent with a failure to shut off protein 100 tivated in the Ensa-depleted extract, yet despite phosphatase(s)+Buffer upon entry into M phase (25). PP2A-B55 CDK1 strong histone H1 kinase activity, the extract failed The+Ensa Gwl-Arpp-Ensa module appears to be active www.sciencemag.org (%) to enter mitosis. in+Ensa humanGwl cells, because reduction of Gwl levels We tested the results of adding thiophos- by RNA interference blocks cells in G2 phase of phorylated Ensa to egg extracts. Supplemental the cell cycle (27). activity Enzyme fig. S4 shows that when extra Ensa was added to It is still difficult to understand the details of interphase extracts supplemented with cyclohex- how gradually accumulating cyclin levels are

Mitotic PPase activity phosphoprotein imide and a small amount of recombinant stable converted into the sharp activation of MPF at the (APC3) cyclin B (not enough0 to induce mitosis by itself), G2-to-M transition. Gwl appears to require acti- 55δ Downloaded from Time phosphorylation events typicalC of mitoticA+C A+B extracts+C vation by MPF (11), and once it is turned on, it were induced when Gwl-thiophosphorylatedRecombinant PP2As Ensa phosphorylates Ensa, which, in turn, switches off was added. Dephosphorylated Ensa or Ensa thio- PP2A-B55d (Fig. 4). This promotes the activa- CDK? Greatwall/Rim1phosphorylated5 by PKA did not show any effect.PKA? Chk1tion? of MPF by increasing phosphorylation of S. cerevisiae These results are all consistent with the idea thatWee1, Myt1, and Cdc25, and it also assists entry Fig. 4. Diagram of how PP2A-B55d is inactivated S. pombe Mochida et al., 2010 ScienceGwl 330 inhibits, p1670 PP2A-B55dby phosphorylating Ensa,into mitosis by reducing the dephosphorylation C. elegans by CDK via the Gwl and Ensa pathway. Pathways in D. melanogasterblue are active in interphase; pathways in red thereby both activating Cdk1 (by inhibiting Wee1of MPF targets. This is an example of a “coherent X. laevis and turning on Cdc25) and allowing Cdk1 to ef- feed-forward loop” (28), because Cdk1 phos- M. musculus denote those active during mitosis. H. sapiens ficiently phosphorylate its target proteins in mitosis phorylates its own activation module in a positive by suppressing the activity of the main opposing feedback loop, it phosphorylates its mitotic target 67 Fig 2 | α-Endosulfine and(fig. ARPP-19 S3A), are indicating Greatwall-dependent that residue inhibitors Ser ofof PP2A Ensa-B55. (proteinA) Schematic phosphatase. diagram of CDK1 and PP2A-B55 activity duringproteins, the cell cycle. and it indirectly inactivates the antago- 15 The patterns of CDK1 andis PP2A crucial-B55 for activity proper are cell-cyclecomplementary control. to each Tyr other; ofCDK1 activityIn this is shown paper, in red we and identify PP2A-B55 Ensa activity and in Arpp-19 green. The phosphorylationnizing protein phosphatase by activating Gwl and status of Apc3/Cdc27 reflectsCdk1 the was ratio dephosphorylated of kinase to phosphatase in activity all three (upper condi- bands indicateas phosphorylation-dependent mitotic hyperphosphorylation). inhibitors (B) Sequence of PP2A- alignmentthe of downstream the Ensa phosphatase inhibitor Ensa. Clear- 15 subfamily from yeast to human.tions, butThree in possible the Ensa-depleted phosphorylation extract, sites are Tyr indicatedre- withB55 arrows.d and The physiological CDK consensus substrates site is found of only Gwl in kinase.Xenopus Ensa,ly, but once is well this system is active, the cell switches conserved in the ARPP-19mained subfamily. dephosphorylated (C) Protein phosphatase for the (PPase) duration assay of using the a modelIn Drosophila CDK substrate,Ensaisrequiredforproperspindle and a catalytic C monomer, A+C dimer or heterotrimercompletely into mitosis in a kind of latch mech- 15 holocomplex containingincubation, B55δ. Ensa phosphorylated whereas in the by controls,Gwl (red bars) Tyr inhibitswas PP2A re- trimericassembly holocomplexes and oocyte that contain maturation; B55δ, but endosulfine- not dimeric or monomericanism. Thus, both Gwl and Ensa are essential for PP2A complexes. Fig 2Cphosphorylated. is a modified version This of figure experiment 2A from Mochida implies et al that[46]. APC3,deficient anaphase-promoting oocytes are unable complex to subunit pass between3; ARPP-19, pro- cyclic-AMP-regulatedthe maintenance of the mitotic state, implying phosphoprotein of 19 kDathe; CDK, main cyclin-dependent effect of not kinase inhibiting; Ensa, PP2A-B55α-endosulfined; isGwl, Greatwall;phase of PKA, first cyclic-AMP-activated meiosis into metaphase protein kinase (24– A26. ). that inhibition of protein phosphatases is critical- in antagonizing Cdk1 phosphorylation of down- Tellingly, these oocytes have high Cdk1 activity ly important for this process. How the return to stream target proteins, rather than on the control yet display little phosphorylation of mitotic sub- interphase is brought about is unclear. When cy- supposed to be involvedof Cdk1 in the activity. control Somehow, of insulin Cdc25secretion or [41]. an equiv- But notstrates, suppressed exactly and as we CDK observed substrates in areXenopus never ex-fully phosphorylated.clins are degraded and Cdk1 levels fall, Gwl and this original idea could not be confirmed and now seems unlikely, A threefold increase in PP2A-B55δ concentration induces a simi- owing to the absence of a secretion-signal sequence in Ensa. In addi- lar phenotype, presumably because the increased PP2A titrates out tion, little Ensa1672 was found in biological membrane fractions17 DECEMBER [42]. endogenous 2010 VOLEnsa 330[15]. Furthermore,SCIENCE thewww.sciencemag.org addition of active—thio- Thus, for nearly 20 years after the identification of these proteins, no phosphorylated—Ensa is enough to induce significant phospho- molecular function had been found. The first evidence for the impor- rylation of CDK substrates even at low levels of cyclin, below those tance of Ensa in cell cycle control came from a study in Drosophila, required for normal mitosis. These observations collectively sug- which has only one gene in this protein family. An RNAi screening gest that even full CDK1 activation is unable to promote entry into using somatic S2 cells identified Endos (the Ensa homologue in mitosis—inactivation of phosphatase(s) is also required. The Gwl Drosophila) as a protein important for mitotic chromosome align- pathway evolved to achieve this seesaw-like relationship. ment and normal spindle length [43]. Drosophila oocytes deficient A word of caution is necessary, however, because not all cell in Endos show high CDK activity with low phosphorylation of CDK divisions seem to depend on the Gwl–Ensa/ARPP-19 system. For substrates, indicating that a lack of Ensa somehow changes the bal- example, although Drosophila that lack Endos are inviable, loss ance between kinase and phosphatase [44]. We and others indepen- of one copy of the twins/aar gene that encodes the B55 subunit of dently discovered that Ensa and ARPP-19 are phosphorylated by PP2A rescues the lethality, although not the female sterility [47]. Gwl at a highly conserved serine residue—Ser 67 in Xenopus Ensa It is difficult to interpret these data with our current understanding (Fig 2B)—becoming potent inhibitors of PP2A-B55δ (Fig 2C; of the pathway. [45,46]). Importantly, this inhibition is highly specific for PP2A-B55δ; other forms of PP2A are unaffected [46]. Although the exact Specificity and regulation of the Ensa/ARPP-19 family sequence that is phosphorylated by Gwl (KYFDSGDYNM) is found Unlike okadaic acid, Ensa is highly specific for the particular species only in these two small proteins, Gwl could have other substrates, as of PP2A that contains the B55δ subunit (Fig 2C) and does not bind to the stringency of its substrate recognition sequence is unknown. other forms of PP2A containing the B56ε, B56γ or B''/PR48 regulatory subunits [46]. Thus, different PP2A holocomplexes are distinctly Reversing the balance of CDK1 and PP2A-B55δ regulated. It is highly probable, however, that other isoforms of B55 Phosphorylation of Ensa/ARPP-19 by Gwl is essential for CDK sub- (α and β) are also targeted by Ensa/ARPP-19 [48,49]. strates to be highly phosphorylated in Xenopus embryonic mitosis. In addition to the Gwl phosphorylation site, Ensa/ARPP-19 fam- In cycling egg extracts that lack Ensa, although Tyr 15 dephospho- ily proteins have another highly conserved phosphorylation site at rylation and full CDK activation occur to the same level as in con- their carboxyl terminus [50]. This site—Ser 109 in Xenopus Ensa— trol extracts (albeit somewhat delayed) [46], PP2A-B55δ activity is seems to be phosphorylated by protein kinases that prefer basic

Different complement of proteins present in

different cell cycle states The Cell Cycle is Co-ordinated by Ubiquitin- dependent Proteolysis

Effectively an interplay between the SCF and the APC/C

SCF = Skp1 + Cullin + F-box protein

APC/C = Anaphase Promoting Complex/Cyclosome Ubiquitination

UBC Ubiquitin Ligase E1 E2 E3 ATP

E1 ADP + Pi E2 E3

Ubq Ubq

Substrate Ubq DESTRUCTION Ubq 26 S Proteosome

Ubq Schematic of RING E3 ligases

UBQ COP9/Signalosome

F-Box/ BTB/ E2 Substrate SOCS Box RING DOC1 Nedd8

Cullin

MammalianF-box cells: proteins 69 F box proteins - bind to Cul 1 Fbw>200 = WD40 BTB domain:proteins Fbw1 -= bind bTrcp1, to Cul Fbw2 3 = bTrcp2, Fbw7 = Cdc4 Fbl >50= leucine SOCS rich box domain: proteins Fbl1 - bind = Skp2 to Cul2/5 Fbx>15 = other DDB1 domains proteins - bind to Cul 4 Nedd8 closes the gap between E2 and substrate

Nedd8

Duda et al., 2008 Cell 134, 995-1006 Interplay between the SCF and the APC/C

• SCFSkp2 is degraded by the APC/C • Allows p27 (Cdk inhibitor) to accumulate in G1 phase

Bashir et al., 2004, Nature 428, 190-193 Ubiquitination: Mitosis

UBC (UBC10/UBC5 + Ube2S) Destruction box proteins E2 E3 Cyclin B Cdc20 Securins

E2 E3 P

Ubq APC/C APC7

APC3 APC10 Apc3 APC6 APC11 APC8

Apc6 Apc8 APC2 APC4 APC5

Substrat e Ubq APC1 Ubq

Ubq

Pines, J. 2011. Nature Reviews Molecular and Cell Biology 12, 427-438 Co-ordinating Mitosis by Proteolysis

Antephase Pro-metaphase Metaphase Anaphase Telophase

NEBD Chromosome Chromatid Cytokinesis Spindle Attachment Separation Disassembly

Spindle checkpoint

Cyclin A Securin Cdc20 Plk1 Aurora A Cyclin B1 UbcH10

Pines, J. 2006, Trends Cell Biol., 16, 55-63 LETTER RESEARCH

abCdc27

Cdc27 RESEARCH LETTER Cdc27

abc Apc10 Apc10

140°Apc10 Cdh1 15° Apc10 Cdh1 Cdh1 Apc2

APC10 and Cdc20 form the Destruction Box receptorApc2 RESEARCH LETTER

APC/CCdh1 APC/C APC/CΔApc10–Cdh1

abcde f

cdApc10 Apc2 IR tail IR tail Cdh1 (C-terminal Cdh1 Cdh1 Apc10 Cdc27 Apc10 Cdc27 Apc10 D-boxCdh1 domain) Apc10 Apc10 Cdh1

Apc10 Cdh1 C box (C-terminal i ii D-box domain) Apc2 Cdh1 APC/CCdh1–Hsl1 APC/CCdh1–D-boxLys ΔApc10–Cdh1APC/CCdh1–KEN-box APC/C APC/C APC/CApc11 Cdh1 Cdh1de fE2~Ub Cullin repeats C box Apc11 Apc10 Apc10 Apc10

Figure 3 Cdh1, Apc10, Apc2 and Apc11 form a substrate-recognition N-terminal to theCdh1 IR motif, is indicated by red spheres. c, Details of the Cdh1 | Cdh1 Cdh1 Cdh1 Cdh1 Cdh1 catalytic module. a, b, Two views of the cryo-EMEM APC/CreconstructionsCdh1–D-boxApc10complex. and Apc10 co-receptorApc10 for D-box. BothApc10 Cdh1 and Apc10 connect to Apc2. The Protein density is represented by a mesh with fitted atomic coordinates of the N terminus of Cdh1, including the C box linking the WD40 domain to Apc2, is Cdh1 b-propeller (modelled), Apc10Figure (ref. 1 22),| Negative-stain Apc2–Apc11 EM reconstructions (modelled on of buddingnot yeast modelled. APC/C show TheCCdh1–Hsl1 first redcomplex arrow (d (i)), APC/C denotesCdh1–D-box the conserved(e) and APC/C loopCdh1–KEN-box (residues Cul4a–Rbx1 of SCF) and Cdc27 (ref.that 26). substrate Only binding the N-terminal to APC/CCdh1b strandinvolves of Cdh1 andHis Apc10. 239 a– toc, Asp Mol- 244)(f). of Lower Apc10 panels implicated in d, e and f inshow D-box details recognition of the structural7, and changes the associatedsecond ecular envelopes of APC/CCdh1 (a), APC/C (b) and APC/CDApc10–Cdh1 (c). with Cdh1 and Apc10 in the presence of substrate compared with the Apc11 bound to the Apc2 C-terminal domain is modelled (orange). The two red arrow (ii) denotes the Lys 162 and ArgCdh1 163 of Apc10 responsible for APC/C Density assigned to Cdh1 and Apc10 is shown in magenta and blue,7 superimposed binary APC/C map represented in mesh. Hsl1 and D-box subunits of Cdc27 are shown in lightrespectively. and dark The green. resolution The of view the APC/C in a showsCdh1 binary the complexaffinity is ,18–20. Two A˚ modelsand KEN-box for a possible peptides fit were of used D-box at saturating to the density concentrations interconnecting to promote Da Fonseca et al., 2011, Nature 470, 274-278 Cdh1 two-fold symmetry axis of Cdc27. Density(Supplementary connecting Fig. 10d).APC/C Cdh1d–fCdh1–Hsl1, Negative-stain to a TPR EM reconstructionsAPC/CCdh1Cdh1–D-box and of APC/ Apc10 arestoichiometric shownAPC/C inCdh1–KEN-box APC/C Supplementary–substrate Fig. ternary 8. d complexes., Schematic of combined superhelix of the Cdc27 dimer is indicated by an arrow. TPR motifs 8 to 10 of catalytic and substrate-recognition module responsible for D-box binding and Cdc27, implicated in IR-tail recognitionTo23, explore are shown the structure in lighter of colours. APC/CCdh1–D-box In b, the insubstrate more detail, ubiquitylation. we Cdh1 WD40 D-box domain is represented into their respective as binding densities to an interface indicates further final residue of Apc10 observed in thecollected crystal cryo-EM structure images (Ser of 256), theApc10 complex 25 residues and determinedbetween its structureApc10 Cdh1 andunassigned Apc10. densityApc10 linking Cdh1 to Apc10 (Fig. 3a, c). Notably, the at ,10 A˚ resolution. The cryo-EM map reproduces the overall features best fit of Apc10 into the cryo-EM map positions a highly conserved Cdh1–D-box 1 715 rationalizes biochemical studiesof demonstrating the APC/C Cdh1 thatmap both generated co-activator from negativelyCdh1librium. stained Consequently, part- loop requiredCdh1 the features for D-box of recognition the H- adjacentN-HSQC to the spectrum density linking are 8,9,11,14–17icles, but with greatly enhanced detail and resolution (Fig. 2 and Apc10 with Cdh1. In contrast, residues on the opposite surface of and core APC/C subunits , specifically Apc10 (refs 5–7, 23), Cdh1–D-boxbest explained as a result of Apc10 adopting multiple conformations7 in contribute to D-box-dependentSupplementary recognition Figs and 6 and processive 7). Similar ubiquity- to the APC/C intermediateternary toApc10 slow exchange that contribute (submillisecond to APC/C interactions to secondare oriented timescales) towards Figure 1 | Negative-staincomplex EM obtained reconstructions from negative-stain of budding yeast EM, APC/C the cryo-EM show reconstruc-CCdh1–Hsl1 complexApc2 (d (Fig.), APC/C 3c).Cdh1–D-box (e) and APC/CCdh1–KEN-box lation. The unassignedthat density substrate binding bridgingtion shows to APC/C Apc10 densityCdh1 andinvolves connecting Cdh1 Cdh1 in Cdh1 and the Apc10. and APC/ Apc10 a–c, Mol-in (Figs solution.(f 2). andLower 3). panels Addition inThesed, e and structural off show a stoichiometric details data revealing of the structural that excess Cdh1 changes and (, associated Apc1040-fold) become of inter- the Cdh1–D-box Cdh1–D-box C complexecular can envelopes be modelledDocking of APC/C the asCdh1 crystala(a D-box), APC/C structure peptide, (b) and of APC/CApc10 indicatingD (refsApc10–Cdh1 13, 22)(c). andD-box thewith modelled peptide Cdh1 and usedconnected Apc10 to in generatethe by presence bridging the of substrate density APC/C compared in the presence with theternary of D-box complex substrates Cdh1 that the binding siteDensity for assigned the D-box to Cdh1 andis shared Apc10 is shown between in magenta the and WD40 blue, resultedsuperimposed in more binary than APC/C 20 changesmap represented in amide in mesh. peak Hsl1 position and D-box or relative respectively. The resolution of the APC/CCdh1 binary complex is ,18–20 A˚ and KEN-box peptides were used at saturating concentrations to promote domain of Cdh1 and(Supplementary the b sandwich Fig. 10d). d– off, Negative-stain Apc10. Cdh1 EMab reconstructions and Apc10 of APC/intensitystoichiometric (Fig. 4). APC/C NMR-basedCdh1–substrate c ternary measurement complexes. of the translational dif- therefore generate a D-box co-receptor (Supplementary Fig. 8). fusion coefficient showed that the NMR-observed species is an Apc10 Although biochemicalTo data explore show the structurethat the of D-box APC/CCdh1–D-box interactsin with more the detail,monomer we Cdh1 WD40 of ,26 domain kDa. into Thus, their the respective changes densities in specific indicates peaks further on addition collected cryo-EM images of the complex and determined11,15 its structure unassigned density linking Cdh1 to Apc10 (Fig. 3a, c). Notably, the conserved surface of the WD40˚ domain of the co-activator , direct of peptide demonstrate that the D-box peptide interacts with mono- at ,10 A resolution. The cryo-EM map reproduces the overall features best fit of Apc10Apc10 into the cryo-EM map positions a highly conserved interactions betweenof D-box the APC/C andCdh1–D-box Apc10 alonemap generated have not from been negatively previously stained part-mericloop Apc10,Cdh1 required altering for D-box14° the recognition chemical150° 7 adjacent environment to the density and/or linking the confor- demonstrated (unpublishedicles, but with data greatly and enhanced ref. 7), possibly detail and owing resolution to (Fig. the 2 andmationalApc10 equilibrium with Cdh1. In of contrast, a subset residues of onits the residues. opposite However, surface of the low weak affinity of isolatedSupplementary Apc10 for Figs D-box. 6 and 7). Similar to the APC/CCdh1–D-box ternaryintensityApc10 and that contribute proportion to APC/C of visible interactions amide7 are peaks oriented made towards sequential We used 1H-15N-heteronuclearcomplex obtained single from negative-stain quantum coherence EM, the cryo-EM (HSQC) reconstruc-assignmentApc2 (Fig. and 3c). full characterization of the D-box binding site on tion shows density connecting Cdh1 and Apc10 (Figs 2 and 3). These structural data revealing that Cdh1 and Apc10 become inter- NMR, a technique suitableDocking thefor crystal detecting structure weak of Apc10 protein–ligand (refs 13, 22) and interac- the modelled20° Apc1090° connected impracticable. by bridging density in the presence of D-box substrates tions, to investigate potential Apc10–D-box interactions. The 1H-15N- To establish whether the peptide-induced changes of the Apc10 HSQC NMR spectrum of Saccharomyces cerevisiae Apc10, shown in NMR spectrum are specifically D-box dependent, we performed a ab c Fig. 4, has a substantial number ofFigure well-dispersed 2 | Cryo-EM reconstruction peaks consistent of budding with yeast APC/CseriesCdh1–D-box of controlreveals the experiments. lattice-like architecture First, of a the different complex. a D-box–c, Three peptide views of the (a complex 19- the Apc10 b-sandwich architecturewith b22similar. However, to views shown the number in Fig. 1. Resolution of visible is ,10 Aresidue˚ (Supplementary peptide Fig. modelled 12c). on S. cerevisiae Clb2 whose sequence identity peaks is approximately half that2|NATURE|VOL000|00MONTH2010 expected for a 221-residue protein, with Cdc13 is confined to the D-box) produced very similar NMR ©2010 MacmillanApc10 Publishers Limited. All rights reserved and the visible peaks have a wide range of intensities. Reduced peakCdh1 spectral changes14° 150° to the Cdc13 D-box (Fig. 4). Second, a mutant number and intensity variation are characteristic of proteins under- D-box Cdc13 peptide resulted in only minor changes in the Apc10 going exchange between different conformational or oligomeric states. NMR spectrum, consistent with greatly reduced binding. Finally, the Spectra recorded with a twofold difference in protein concentration Hsl1 KEN-box peptide that, from the APC/CCdh1–KEN-box EM analysis, showed no change in position or shape of any dispersed peak, indi- does not bridge Cdh1 and Apc10, resulted in an essentially identical 20° 90° cating that there is no sensitivity to any possible oligomerization equi- spectrum to that of the apoprotein, with none of the changes seen for

00 MONTH 2010 | VOL 000 | NATURE | 3 Figure 2 | Cryo-EM reconstruction©2010 of buddingMacmillan yeast APC/CPublishersCdh1–D-box Limited.reveals theAll lattice-likerights reserved architecture of the complex. a–c, Three views of the complex with b similar to views shown in Fig. 1. Resolution is ,10 A˚ (Supplementary Fig. 12c).

The APC/C-Cdc20 degrades Cyclin B1 when all chromosomes are attached to the spindle Exit from mitosis is coupled to re-licensing of origins of replication The APC/C is inactivated before cells begin S phase - allows mitotic cyclins to reaccumulate APC/C speciﬁcity: two co-activators

Cdc20 Cdh1 Required for correct G1 phase & to degrade Aurora Essential for Mitosis kinases

Regulated by spindle checkpoint Regulated by phosphorylation (by CDKs)

proteolysis by Cdh1 Rca1/Emi1

Proliferating cells Somatic & Differentiated cells (brain & trophectoderm)

Yu, H. 2007, Mol Cell., 27, 3-16; Garci-Higuera et al., 2008, Nature Cell Biology, 10, 802-811 Floyd et al., 2009 Curr Biol., 18, 1649-1658 Cdc20 and Cks target Cyclin A-Cdk to APC/C

Cyclin A outcompetes the SAC proteins Cdc20 for Cdc20 Cyclin

Cdk

Cks

APC/C

Wolthuis et al., 2008, Mol Cell 30, 290-302; Di Fiore & Pines, 2010, J. Cell Biol 190, 501-509 Completion: Timing and Checkpoints

Chromosome Separation

DNA replication Completion: Timing

Budding yeast mitosis (at least under laboratory conditions)

Embryonic cell cycles

Xenopus (all you need is cyclin B)

Drosophila

Problem: High error rate Inﬂexible Completion: Checkpoints

First deﬁned by Weinert and Hartwell, 1989, Science 246, 629.

“The events of the cell cycle .. are ordered into dependent pathways in which the initiation of late events depends on the completion of early events.

Some dependencies can be relieved by mutation ….suggesting that the dependency is due to a control mechanism and not an intrinsic feature of the events themselves. Control mechanisms enforcing dependency in the cell cycle are here called checkpoints.” Checkpoints:

S phase checkpoint M G2 Checkpoints Ds breaks UV

Morphogenesis Checkpoint S Prophase Checkpoint

Spindle Checkpoint Checkpoints

Think about the biology

– DNA damage: budding yeast arrest in mitosis ﬁssion yeast and animal cells in G2

What should be the phosphorylation state of Cdk1?

What should be the targets of the checkpoint? Checkpoints

• Budding yeast do use Cdk1 Y18 phosphorylation

• Part of the morphogenesis checkpoint to prevent budding in inappropriate conditions

• Major regulation is on the stability of Swe1p

• Degradation of Swe1 accompanied by relocalisation to bud neck via binding to Hsl7p

McMillan et al., 2002, Mol. Biol. Cell., 13, p3560-75 Summary

The cell cycle is driven by alternation of high and low Cdk activity

Robust switches are driven by ultrasensitivity and phosphatase feedback loops

Alternation in Cyclin-Cdk activity is underpinned by proteolysis

In somatic cells checkpoints ensure that the switch is not thrown until previous stage is complete (embryos often rely on timing)