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Summary: Put Out More Flags T. HUNT

ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, England

Almost 50 years went by before a Cold Spring Harbor Symposium devoted to the cell cycle was achieved. Previously, the Symposium that came closest to discussing "cycles" was arguably the 1960 meeting on Biological Clocks (CSH Symp. Quant. Biol. 25 [1961]), with articles on topics like Lunar Periodicity (p. 491) and Time-Compensated Sun Orientation in Bees (p. 371). Why did it take so long to get to cell growth and cell division? The short answer is: not enough was known. In their magisterial General Conclusions of the groundbreaking 26th Symposium on Cellular Regulatory Mechanisms in 1961, Monod and Jacob wrote (p. 392): "Little has been said during this conference of the mechanisms which control cell division," a telling phrase, in that they did not use the word "cycle," and true. They alluded to the pioneering studies of Rapkine (1931) and Mazia (1959) (the - S H cycle in mitosis and inhibition of cleavage in sea urchin eggs by mercaptoethanol). Monod and Jacob presumed that "molecular conversions" were involved in, or govern, the cell cycle, but studies on thiol interconversions in mitotic cells hardly pointed toward the successes of the late 1980s, and with hindsight, we can see that 30 years ago, even the most advanced thinking about the cell cycle was apt to be off-beam. The only faint echo of the importance of - S H groups today comes perhaps from the widespread use of glutathione S-transferase as an affinity tag for studying protein:protein interactions (Solomon et al.; Chittenden et al.). So until very recently, there existed only scattered clues about the control of the cell cycle. Good clues, many of them, but scattered. To do the pioneers justice, however, it should be said that Mazia's long review in The Cell (1961) is still worth reading, and despite recent successes, especially in putting flesh as it were on his famous pistol, most of the problems discussed there remain with us today. The 1991 Symposium was an outstanding example of what a scientific meeting should be. People came together at the right time in the right spirit, and streams of exciting discoveries were presented and discussed (discussions used to be printed in earlier Symposium volumes, which were very helpful for the baffled student) in the beautifully appointed new Grace auditorium and at the crowded poster sessions. There was a feeling of celebration in the air, brilliantly captured

All authors cited here without dates refer to papers in this volume.

by Mike Bishop in his Dorcas-Cummings lecture, which rehearsed the extraordinary progress that biology has made since Robert Hooke's first sight of cells over 300 years ago. Thanks are due to Drs. Beach, Stillman, and Watson for their sense of timing and choice of participants, and also especially to those whose tireless work behind the scenes allowed the science to shine. Only the Blackford lawn was missing. The recent growth of the cell cycle field has been amazing to watch, and exhilarating to ride. Back in 1982, when I was first drawn in, very few people were interested; indeed, to take an interest was to risk being thought a nut. There were yeast people of course, such as Lee Hartwell, Murdoch Mitchison, Paul Nurse, and their followers, and frog people like Yoshio Masui, John Gerhart, Marc Kirschner, and Jim Mailer; the Starfish Gangs, one French and the other Japanese, and a few--very f e w - meetings were organized under the aegis of the European Cell Cycle Group, which contained a suspiciously chronobiological wing. The mitosis and motility crowd approached the cell cycle in a different spirit, mainly concerned with structure and movement, rather than control. In fact, of those I talked with or listened to in the early days, only Marc Kirschner and John Gerhart seemed to see things much as we now see them, except back then, embryo people were pretty ignorant (downright dismissive in certain cases) of yeast and yeast genetics. It was very odd that there were so few meetings to discuss this central and important topic in cell biology, and when in the autumn of 1986, it was suggested to Jim Watson that Cold Spring Harbor might bring some people together, he abruptly changed the subject. This was probably correct at the time, but just 2 years later, in the spring of 1988, a highly successful gettogether of about 40 participants took place at the Banbury Center, where cdc2 and cyclin people first met properly and the miraculous properties of p13 Sue1 Sepharose came to light. The CLN1,2, and 3 genes had recently been "discovered," as had the cyclin A gene of Drosophila and its mutant phenotype. It was perfectly clear, although it was not stated in public, that the maturation-promoting factor (MPF) which Fred Lohka and Jim Maller had purified from Xenopus eggs about 2 years earlier contained cdc2 and cyclin. In September of 1988, about twice as many people met in Roscoff, France (the first CNRS-sponsored Jacques Monod Cell Cycle Conference), by which time MPF, cdc2, and cyclin were center-stage. Phase one was complete. Genetics and biochemistry had met, and the relation-

Cold Spring Harbor Symposia on Quantitative Biology, Volume LVI. 9 1991 C01dSpring Harbor Laboratory Press 0-87969-061-5/91 $3.00

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ship was exciting, if stormy and fraught with misunderstandings. In the Spring of 1990, just over 200 people met briefly at a meeting in Airlie House, Virginia, now under the highly respectable banner of the ASCB and EMBO, and a year later in Cold Spring Harbor, the numbers had doubled again. This mushroom growth is not surprising, considering the importance of cell cycle control. Yet one of the most frustrating things from a cell cycle perspective is the continuing tenuous connection between the hugely successful oncogene field and cell cycle control in the strict sense. I thought we were going to see bridges built at this Symposium, but they are not quite there yet. Certainly not yet strong enough to accommodate the waiting armies on the other side.

What Is the Cell Cycle? Hardly anyone at the meeting bothered, or had time, to consider what was meant by the term "cell cycle." This is not an entirely empty or purely semantic point, for misunderstandings often stem from disagreement on this question, whose two (often implicit) views Murray and Kirschner (1989) called "Dominoes and Clocks." Thus, Murdoch Mitchison (1970) wrote in his book that the cell cycle was "the fundamental unit of time at the cellular level," and for him still, as he said in his talk, "timing is the essence." This is certainly one way of looking at things, but how useful is it? Timing implies a clock that runs on an oscillator of fixed period. You can reset the clock, of course, but I do not think this is what usually controls cell division. There is no oscillator, no biochemical device that emits regular pulses that the cell cycle control machinery counts. Cell cycle timing is like scientists' timing: 'Tll come home when the gel's run," and not 'TI1 be home by 8." (This is why it is important to be very careful talking about time, whether in discussing the cell cycle or dinner.) But it is hard to avoid using phrases such as "this determines the timing of entry into mitosis," because this is what you usually actually measure. A different, and I think more apt, description of the cell cycle was provided by Murray and Kirschner, who wrote in their review, "The cell cycle is the set of events responsible for the duplication of the cell." This is laconic, however, and I like the way Helena Curtis put it in her textbook, Biology (4th Edition, 1983): Dividing cells pass through a regular sequence of cell growth and division, known as the cell cycle. The cycle consists of five major phases: G 1, S, G 2, mitosis, and cytokinesis. Completion of the cycle requires varying periods of time from a few hours to several days, depending on both the type of cell and external factors, such as temperature or avaliable nutrients." This apt and modest paragraph is a good description and does not impose a model. The field has been a province for modelers, and I do not think they are very useful to us, yet. The question is: What more can we now say?

Cell Cycle Progression Is the Default Pathway and Checkpoints Enforce Order Understanding the control of the cell cycle is really very simple. It hangs on the answer to the question: What principles does the cell use to establish an ordered pathway of events? (Hartwell and Weinert 1989). The main answer, clearly enunciated by Hartwell and Weinert, is checkpoints. But, if checkpoints are the key, how many checkpoints are there? And is it really true that there are no intrinsically ordered events in cell cycles of the everyday kind familiar to humans (i.e., underpants go on before trousers)? In their respective chapters, Hartwell (Brown et al.) and Murray discuss two of the best known checkpoints, both of which are concerned with monitoring the internal state of cellular systems. The RAD9 gene is part of the DNA-damage-checking system, and the MAD2 gene is part o f the mechanism that checks the integrity of the mitotic spindle. As these authors point out, the logic of this kind of control means that mutations in this class of gene are recessive, and loss-of-function mutants undergo inappropriate, because unchecked, cell cycle progression, with disastrous consequences only when something is wrong. It is strange how variable such checking seems to be (Schimke et al.). It is important to remember that there are no absolute rules about what happens in the cell cycle. M phase can follow immediately after M phase, as it does in meiosis. S phase can follow S phase with no chromosome segregation, as happens in polyploid and polytene cells. Some cells are very large, some are very small, and some divide at a very narrowly defined size. Checkpoints are plug-in modules that can be added onto the basic cell cycle engine to regulate its progress. As we shall see later, genetic analysis in yeast suggests that the default state of the engine is to keep running. Not all checks are internal, of course, and according to this view of cell cycle progression, growth factors and mating pheromones represent external conditions that can provide a cell with instructions to proceed or halt. Nutrient supply may act the same way, although in this case, it makes more sense for cells to maintain an inventory of their internal stores of carbon, nitrogen, and phosphorus and thereby forecast materials and energy futures. In yeast, sensing both food and sex Are we fertilized? Are the chromosomes lined up? Is there any progesterone?

Are the chromosomes intact?. Are chromosomes completely replicated?

finish (anaphase) Are we big enough? Are food reserves adequate? What signals are we getting from the neighbours? start

(restriction point) Figure 1. Some well-known checkpoints in the cell cycle.

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SUMMARY involves ras-related GTP-binding proteins coupled in one way or another to protein kinases (Cross; Herskowitz et al.). The details of the signal transduction pathways involved in cell cycle regulation will probably prove as diverse as those found in other spheres of biology, and no different in principle. Figure 1 shows a conventional cell cycle with just four phases and some simple examples of known checkpoints. MPF and the Control of Mitosis

The particulars of cell cycle control are best worked out for entry into mitosis. MPF is what causes frog oocytes to mature, i.e., to pass from G 2 (the progesterone checkpoint) into M phase of first meiosis and proceed to a second cell cycle arrest at meiosis II metaphase (the fertilization checkpoint) (Masui and Markert 1971). This is a complex process that takes much longer in amphibian oocytes than it does in clams and starfish (hours as opposed to minutes) and mixes elements of pure control over entry into M phase with elements of the kind of resting-to-growing transition more familiar in the context of serum-starved or contact-inhibited tissue-culture cells. Probably, MPF also underlies the classic cell fusion experiments of Rao and Johnson (1970), which demonstrated the dominance of the mitotic state over all other cell cycle states. It is often said, both in speech and in writing, that MPF is cdc2 kinase. This is unfortunate and potentially misleading because MPF is defined by an assay that responds to many factors besides "cdc2 kinase." They include cdc25, a phosphatase (indeed, I suppose that if cdc25 had been tested first, it would have been MPF); okadaic acid, a phosphatase inhibitor; activated ras genes; activated transmembrane tyrosine kinases and activators of such kinases, like insulin; the serine/ threonine kinase, c-mos and v-mos; and cyclins A and B. Several of these even meet the criterion that they will act in the presence of protein synthesis inhibitors, and although many of them probably act by turning on the protein kinase activity of endogenous cyclin B/cdc2 complexes, this is by no means completely proven. And although MPF itself certainly contains active cyclin B/ cdc2, and such complexes do cause oocyte maturation, other factors contribute to the activity of the crude cytoplasm that Masui originally injected into oocytes. Indeed, in 1980, Wu and Gerhart (1980) showed compelling evidence that MPF contained more than a single component and demonstrated that it was easily inactivated unless certain precautions, such as inclusion of ATP in the buffer, were taken. The biochemical basis of these observations is still not clear. Understanding how the state of a cell changes so dramatically from interphase to mitosis and how the state is maintained for just as long as it needs to allow the spindle to form and align the chromosomes is by no means complete. In particular, what component of MPF needs to be phosphorylated for retention of activity is not yet clearly defined, although at least one explanation appears to

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lie in the requirement for phosphorylation of Thr-161 on p34 cdc2 itself (Ducommun et al. 1991; Gould et al. 1991; Krek and Nigg 1991; Norbury et al. 1991; Solomon et al. ; Nigg et al.; Brambilla et al.). There are, moreover, conditions when cdc2 kinase is turned on full, yet some cells do not enter mitosis. This has been most clearly shown by Osmani et al. (this volume), whose nimA kinase in Aspergillus seems to be absolutely required, presumably in parallel to cdc2 (Osmani et al. 1991). There is no doubt that the activity of cdc2 is required for the cell to pass from interphase into mitosis, but according to my reading, this meeting announced the end of the age of innocence in which all cell cycle control boiled down to questions of how the kinase activity cdc2 is turned on and off, representing the "master regulator." Several other hints of this were apparent. For example, Nigg et al. did experiments in which cdc2 was artificially activated by mutations in Thr-14 and Tyr-15. The activated kinase caused a subset of mitotic events to occur in the recipient cells. The cells rounded up and lost their nuclear envelopes but did not appear to form spindles or condensed chromosomes of the sort seen in classic examples of premature chromosome condensation. Nasmyth et al. reported that the mitotic activity of CDC28 is still high in cdc15 mutants at the nonpermissive temperature, which have undergone anaphase. My suspicion is that reports from several laboratories of the high activity of MAP kinase during meiotic maturation (Ferrell et al. 1991; Gotoh et al. 1991; Ruderman et al.) are significant and that this kinase may play a role in keeping the cell in M phase between the two meiotic divisions. The kinase activity of cyclin B/cdc2 transiently falls at this time, yet the chromosomes stay condensed and nuclei fail to reform. The sequence of MAP kinase shows that it is a close relative of the cdc2 family, and like cdc2, it apparently phosphorylates serines to the left of prolines. There is also an intriguing reciprocal relationship between the tyrosine phosphorylation of the two kinds of kinases; cdc2 is turned o f f by phosphorylation on Y-15, whereas MAP kinase is turned on by phosphorylation of a tyrosine residue whose position in the sequence of MAP kinase is probably homologous to T-161 (Payne et al. 1991). Finally, it is important to recall that although genetics identifies cdc2 as a gene that is necessary for mitosis, it cannot speak to its sufficiency. It would be quite wrong to belittle the importance of cdc2, however. Hardly a single speaker at the meeting failed to mention this essential gene and its protein product, with good reason. We now understand a good deal of how it is turned on and off, although there is still much to learn. Both cdc2 and its close relative cdk2 need a companion subunit, a cyclin, for activity during M phase. The activity of the cyclin/cdc2 complex requires there to be a serine or threonine at position 161 of p34Cdc2; alanine or valine at this position is lethal (although curiously, if Brambilla et al. are correct in the claim that phosphorylation of Thr-161 is required for cyclin binding, it is

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odd that they also find that the valine mutant is a dominant negative mutation when introduced into S. pombe). Good progress is being made in producing active cyclins and cdc2 in bacteria and in insect cells with baculovirus vectors (Maller et al.; Piwnica-Worms et al.). It will be very helpful to identify protein kinases that are required to activate the binding and the kinase activity of cyclin/cdc2, because these appear to be required for activity, and if large amounts of this family of protein kinases are to be produced for biochemical and structural studies, it will be important to identify and purify them. Tremendous progress has been made in fathoming the details of the proteins encoded by the important wee1 § and cdc25 § genes (Millar et al.; Piwnica-Worms et al.; Solomon et al.; Kumagai and Dunphy). Only 1 year ago, it was legitimate to question their normal role in the cell cycle, considering that when both genes were deleted in S. pombe, the cell cycle was not much affected and the yeast grew normally. We now know that these genes are redundant, with mikl covering for wee1 (Lundgren et al. 1991) and stfl apparently backing up cdc25 (Hudson et al.). wee1 is a mysteriously specific tyrosine kinase that appears from its sequence to be a serine/threonine kinase. Its only known target is cyclin/cdc2, but it belongs to a family containing other genes that play roles in specific cell cycle control, like MCK1, which controls sporulation and has a role in the function of kinetochores in budding yeast (Sikorski et al.). Likewise, cdc25 apparently encodes a superspecific tyrosine phosphatase that only seems to work on cyclin/cdc2 under normal circumstances and that only revealed its true homology with phosphatases as a result of the discovery of novel tyrosine phosphatases in the plague bacillus and vaccinia virus (Kumagai and Dunphy; Solomon et al.). A key cysteine residue at the active center of these enzymes finds it counterpart in cdc25, and when mutated, both tyrosine phosphatase activity and the ability to complement a cdc25- strain of yeast are lost. Although the activities of both weel and cdc25 are probably regulated, our understanding of that regulation is primitive, weel is thought to be involved in the sensing of cell size and nutritional status, and nothing presented at the meeting altered or elaborated on that idea (Millar et al.; Piwnica-Worms et al.; Hudson et al.). On the other hand, cdc25 is thought to receive input from the checkpoints that determine whether D N A replication is complete, and D N A structure undamaged, but the nature of the transduction of these signals and the way they impinge on cdc25 remain a topic for further investigation (Enoch et al.). Kumagai and Dunphy found that cdc25 purified from bacteria could activate the inactive cyclin B2/cdc2 complexes found in stage VI Xenopus oocytes and, furthermore, that cdc25 itself undergoes a large mobility shift due to phosphorylation during oocyte maturation. It is tempting to interpret these findings in terms of the wellknown, but so far unexplained, positive feedback loop that seems to be involved in the activation of MPF. If

active

inactive

0

0

15

161

o

1

15

161

ma,

kinase~**%

~1~ 1

o

15

161

1

1

15

161

inactive

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Figure 2. Phosphorylation can activate and inactivate cdc2. A zero "0" indicates a nonphosphorylated residue, and a "1" denotes phosphorylation at that site. cdc25 turns on cdc2, and cdc2 phosphorylates and activates cdc25, we may not have to look further. But this is going much further than what the authors of these studies would claim. Studies from the Kirschner (Solomon et al.) and Newport (Milarksi et al.) laboratories indicate that the cdc2 Tyr-15 phosphatase shows increased activity in mitosis and that the tyrosine kinase correspondingly shows reduced activity. It has yet to be demonstrated, however, that the activities found in the Xenopus extracts truly represent weel and cdc25. One can be confident that some beautiful tales of regulation will be told, however, once the systems can be isolated and the components become available in pure and active form. The idea is that the activity of cdc2 kinase can be regulated in a way that sensitively reflects a large number of cellular inputs, yet once a decision is made, activation of the kinase is irrevocable. Mitosis rarely, if ever, stutters or peters out, and the dominance of the mitotic state similarly implies the existence of molecular latch mechanisms that serve to prevent chatter as systems pass through thresholds from one state to another. These are the very essence of cell cycle controis, and it will be very interesting to learn how such regulatory circuits are constructed. I have attempted a simplified summary of the states of cdc2 in Figure 2. What Does cdc2 Actually Do?

It is not too clear what cdc2 does, once its kinase activity is turned on. Nigg (Nigg et al.) and Goldman (Goldman et al.) describe how this enzyme can phosphorylate certain members of the intermediate filament family and cause their disassembly; in which case, the evidence is reasonably strong that cdc2 does most of the dirty work itself. But entering mitosis involves considerably more than just breaking down nuclear envelopes, and the really crucial question is: How many substrates change their phosphorylation state during mitosis, and by how much does the phosphorylation state of each member of this class have to change to ensure the orderly progression of events that so profoundly alter the architecture of the cell in mitosis, and so beautifully orchestrate the movement of the chromo-

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SUMMARY so qaes and spindles? It is conceivable that mitosis could be done at the command of just one protein kinase if it we,'e a master switch that coordinated the activity of a series of "slave" kinases. But opinion seems to oscillate between that idea and the extreme alternative that phosphorylating almost any serine in the sequence -SP(the predominant local preference of the mitotic kinase) is what (and all) it takes to alter the state of a cell from interphase to mitosis. Cyclins Target cdc2 to Cellular Substructures

A helpful pointer came from Kellogg et al. that cyclins A and B are both among the proteins bound to mierotubule and MAP affinity columns. These biochemical observations agree with cytological studies (Alfa et al.; Glover ct al.; Pines and Hunter). Given that vertebrate cells contain several kinds of cyclin, and several new relatives of p34 cdc2itself (Meyerson et al.), higher eukaryotes may contain many different, kinds of cdc2 kinases, each one specific for a different set of substrates and each with its own mechanism for timing its activation and inactivation. The idea of targeting protein kinases to substratcs via a rcgulatory subunit that can make specific protein:protein interactions is familiar from studies of cAMP-dependcnt protein kinase, where it seems that the RII subunits show strong binding to MAPs (Carr et al. 1991). There is a difference, in that cyclins are generally supposed to be positive activators of cdc2, whereas the R subunit of cAMPdependent protein kinase is a repressor that can be turned off by cAMP. But the principle seems superficially similar; protein kinases are generally bound tightly to their substrates and can be turned on and off by specific signals. At first sight, this view seems to be contradicted by the ease with which all kinds of mammalian cyclins can substitute for CLN-deficient strains of budding yeast. It is instructive that mitotic yeast cyclins appear to be incapable of performing this function (Reed et al.). We suspect that the relatively nonconserved amino termini of cyclins contain a variety of protein targeting sequences. When these sequences are completely inappropriate, as they may well be in yeast, the heterologous cyclins presumably generate a kind of "plain vanilla" cdc2 kinase, which does an adequate, and under the circumstances, life-saving job of phosphorylating appropriate cellular targets. A clear implication of this analysis is that the homologous yeast B-type cyclins contain features that prevent them from activating Cdc28 at the wrong time in the wrong place. RCCI and a G-protein Check for When to Condense Chromosomes

Microtubules and intermediate filaments are not the only cellular structures that change their architecture in mitosis. Chromosome condensation, which gave mitosis its name more than a century ago, is still exceedingly obscure. For a long time, histone H1 phosphorylation (and other modifications of histones) has been sus-

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pected to be important in the process, but details are largely absent, and the apparent lack of histone H1 in yeast and cleaving frog eggs, despite their condensed chromosomes, means that it is important to keep looking elsewhere or at least to keep an open mind on this question. A good assay system that condenses added chromatin into chromosomes is needed, which the frog egg extract certainly provides; it condenses sperm chromosomes very nicely. Whether it will be possible to dissect this biochemically remains to be seen. Here is an area where yeast are not so helpful, because their chromosomes are too small to be seen under ordinary microscopes. Recently, studies of RCC1 have shed light on this area. When cells bearing temperature-sensitive mutants of this gene are incubated at the nonpermissive temperature, the RCC1 protein is rapidly proteolyzed, and, if the cells were in S-phase, premature chromosome condensation ensues (Seino et al.). Does this mean that RCC1 is a regulator of chromosome condensation or is it simply a component of chromatin, or both? When a frog cell-free system is depleted of RCC1, D N A synthesis can no longer occur (Milarski et al.). The plot thickened with the discovery by Matsumoto and Beach that the fission yeast equivalent of RCC1 is encoded by a gene (piml) characterized by premature entry into mitosis at the nonpermissive temperature. These mutants were rescued by overexpression of a gene called spil, which encodes a small GTPbinding protein highly homologous to human polypeptide TC4, and it was recently reported that RCC1 displays GTP exchange activity for its companion G protein, which is indeed TC4 (Bischoff and Ponstingl 1991). These are exciting discoveries that strengthen the conviction that G proteins will be widely used in cell cycle checkpoint control. This is familiar territory from the interlocking cycles that comprise protein synthesis. Here, a G protein is used to put tRNA into the ribosome, and if the anticodon matches, the GTP is hydrolyzed. Another G protein is used to move the ribosome along the mRNA, and when this is successfully completed, the GTP is hydrolyzed. It is not hard to see how this kind of mechanism could be coupled to sensors that determine whether it is appropriate to proceed to a new cell cycle transition. RCC1 would act as some sort of monitor of the state of chromatin, for example, and when active, it would keep Spil loaded with GTP. If RCC1 is lost by mutation or turned off by the completion of replication, then Spil would be converted to the G D P state. This change is transmitted to the cdc2 kinase activation machinery, and mitosis ensues, provided the level of cyclin B (cdcl3) is adequate. At present, this is hypothetical, but from a biochemical perspective, it feels like the right way to think. A similar thought is spelled out in more detail by O'Farrell and L6opold, whose detailed sequence analysis of the cyclin protein family led them to point out a partial match between conserved motifs in cyclin with a region in the ras gene family. The inhibition of cell cycle progression by mating pheromones in yeast contains a

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G protein in the pathway, which transmits an activating signal to protein kinases that somehow inactivate G~ cyclin/Cdc28 function_ Loss of function mutations in this pathway all lead to sterility because the mating checkpoint is removed and cells cannot prepare themselves for conjugation. This kind of evidence suggests that the default pathway is cell cycle progression and that stopping the cycle requires flags and checkpoint modules.

Membrane Traffic Stops in M Phase Changes in intracellular membrane traffic that occur during mitosis are hardly mentioned at all in this volume. Like the nuclear envelope, the Golgi apparatus breaks down from sheets to vesicles during M phase. Addition of cdc2 kinase to cell-free systems can initiate this process (Tuomikoski et al. 1989). Membrane trafficking seems to involve a plethora of small GTPbinding proteins, some of which are substrates for the cdc2 mitotic kinase (Kinsella and Maltese 1991). Here again, GTP-binding proteins appear to be intimately involved in the control of cell architecture during mitosis, although as Henry Bourne (1988) pointed out in a previous summary, one must be careful to distinguish the protein-synthesis-type G proteins (which may well be used for making sure vesicles are being delivered to the right place) from the aflT-style G proteins that are found in mammalian and yeast extracellular signaling pathways, which are used to flag states of molecular affairs, such as receptor occupancy, or maybe even cellular states, such as bare-store cupboards in the case of cdc25 in budding yeast.

Returning to Interphase: (1) Killing the Kinase Little was said all week about getting out of mitosis and back into interphase, nor will the assiduous reader of this volume be much further enlightened. It is generally believed that the kinase activity of cdc2 is normally turned off by destruction of its companion cyclin subunit and that this destruction is triggered and catalyzed in a special way (Solomon et al.; Ruderman et al.). Details of both trigger and mechanism are sparse. According to Glotzer et al. (1991), proteolysis is preceded by the ubiquitination of cyclin, but we do not yet know how this is regulated, and few (if any) mutations in yeast that affect the process are known, unless the M A D mutants described by Murray are counted (which should be). This is another case of checkpoint control, where normally, misplaced chromosomes inhibit anaphase onset, which requires cyclin proteolysis. In the mutant, this inhibition is weak or absent and cells proceed recklessly through mitosis back into interphase. Parenthetically, I note CDC34, which encodes a ubiquitin ligase that seems to have something to do after START before the onset of D N A synthesis in budding yeast. Ubiquitin is probably a multipurpose flag, which does not necessarily tag a protein for destruction (Finlay and Chau 1991), but we should prob-

ably be on the lookout for other cases of highly specific, regulated proteolysis. There is nothing so final as death. In any case, much more work is required to understand cyclin destruction. The way forward seems to be clear; mutations that affect the process are needed, and affinity chromatography with "destruction domains" would probably be rewarding. The stability of the CLN proteins is also a matter for serious attention; their carboxy-terminal PEST domains probably contain protein motifs that permit accurate regulation of their intracellular concentrations, and finer dissection should prove interesting. Returning to Interphase: (2) The Role of Phosphatases If kinases get cells into mitosis, what enzymes dephosphorylate the mitotic SP sites to get cells back to interphase? Are the timing and order of dephosphorylation of the substrates important? There is some disagreement even as to the identity of the enzyme(s) responsible, with the experts Cohen (Sola et al. 1991) and Yanagida (Kinoshita et al.) on opposite sides of the fence. I certainly would not put money on the issue; having attended two phosphatase meetings as a curious observer, the message I take home is of the probable, but poorly understood, existence of some pretty fancy targeted and regulated protein machines. Unfortunately, phosphatases are technically much more difficult to study than kinases, because of the problems in making appropriate substrates, and genetics may well come to the rescue. Thus, Sutton et al. found that there probably exist protein or proteins that have to be dephosphorylated in G~ for cell cycle progression. It is difficult to interpret these findings or the often interesting results of even very specific inhibitors like okadaic acid on cell cycle events because the cell cycle regulators themselves are regulated by reversible phosphorylation. The results of mutant and inhibitor studies are potentially equally ambiguous, in the sense that it is hard to tell if the inhibition at a particular stage of the cell cycle is ascribable to failure to undo a mitotic phosphorylation pattern or to failure of a regulator (mitotic or START kinases, for example) to switch from one form to the next. The complexity of the network is daunting.

The Mitotic Paradigm Does Not Easily Apply to START There nevertheless is a clear general idea of how the G2/M transition is controlled. When a certain set of proteins is phosphorylated, the cell enters mitosis. Dephosphorylation of the same set of proteins returns the cell to interphase. So, the question is: How does this apply to other cell cycle transitions and, in particular, how does it apply to the G1/S transition or START? Few would seriously suggest that turning on the kinase activity of Cdc28 (for instance) starts up D N A synthesis and turning it off stops D N A replication. Put

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SUMMARY like that, it sounds very silly. Indeed, in that framework, the mitotic paradigm clearly does not apply to DNA synthesis. As far as we know, it is not necessary to maintain cdc2/Cdc28 or a related kinase activity to keep replication running. Thus, there are problems, serious problems, in understanding what most people would regard as the most important cell cycle transition (although as the meeting showed, serious fun besides). First, what exactly is START, and what is its biochemical basis? Like most of the best ideas in cell cycle description and analysis, we owe the concept of START to Hartwell, one of the few real geniuses among a crowd of pretty clever people (Hartwell et al. 1974). As I understand it, START is a point in the cell cycle. It is the point when cells arrest cell cycle progression in response to nutrient starvation, to mating factors, to elevated temperatures in Cdc28 temperature-sensitive strains of yeast, and also to cell size, especially in daughter cells. In mutant strains, START defines the point after which cell cycle progression is no longer temperature sensitive. This shows how thin the ice is when we try to apply the mitotic paradigm, speaking (as some have done) of SPF (S-phase-promoting factor) to parallel MPF; for taken at face value, it means that S-phase progression as such does not require active cdc2 or Cdc28. In the past, arguments have raged about whether the Cdc28 gene product is only required to pass START and is not needed for mitosis in budding yeast. This led to discussions of what constitutes mitosis in budding and fission yeast, and more heat than light. One mutant allele of Cdc28 called Cdc28-1N arrests budding yeast in M phase and can be rescued by expression of B-type cyclins on high-copy-number plasmids (Surana et al. 1991). Thus, Cdc28 is clearly needed for both START and mitosis in both kinds of yeast, and there is no further need for argument (for a useful comparison of cell cycle control in two kinds of yeast, see Forsburg and Nurse 1991a). However, the failure of the majority of mutant alleles of Cdc28 to arrest either in S phase or in mitosis does not allow one to conclude that Cdc28 is not required during S phase. For example, we could say that START marks the point in the cell cycle when Cdc28 becomes stabilized against thermal denaturation, and this coincides with the cell cycle arrest point in response to a-factor or nutrient starvation. These considerations raise the question of how it is that the same protein kinase can promote two quite different cell cycle transitions, a point that Murray discusses in some detail in this volume. In addition, some cellular change of state must occur upon entry into S phase, such that origins of DNA replication "fire" after START but not before it, although they do not all fire at once. The molecular and biochemical nature of this change is still unknown. Furthermore, as is discussed by Ferguson et al., not all origins of replication are used. This may be a result of the block to reinitiation that somehow cancels unfiredbut-replicated potential origins or failure of the particular origin to receive the blessing of the hypothetical

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"licensing factor" in that cell cycle (Laskey et al.). This is an important regulatory feature of chromosome replication that has yet to receive molecular explanation, although Hennessy and Botstein showed that the protein specified by the CDC46 gene, which is thought to be required for the initiation of DNA synthesis in budding yeast, had exactly the properties originally proposed by Blow and Laskey (1988) for their factor.

Thresholds, Switches, and Bootstraps With these thoughts in mind, we next ask: When/s Cdc28 activity required, and what does Cdc28 kinase do, so to speak? Thanks to the yeast people (see Cross; Tyers et al.; Nasmyth et al.; Herskowitz et al.; and Elion et al.), there are fresh clues. At least one of three so-called START or G 1 cyclins, CLN1, 2, and 3, is required, and the complexes between Cdc28 and these polypeptides really do possess protein kinase activity, albeit not very much (the CLN3-associated kinase is especially weedy). Everyone seems also to agree that CLN1 and 2 normally do the real work and that probably, the normal role of CLN3 is to activate the other two. Yet, somehow, CLN3 can do the job in strains lacking both CLNI and 2, hinting at the existence of an as yet unknown CLN4. The activation of CLN1 and 2 needs a pair of transcription factors, SWI4 and SWI6, whose activity in turn depends on Cdc28, presumably because they (or something they interact with) need to be phosphorylated by Cdc28. But, if the transcription of CLN genes needs active Cdc28, and Cdc28 needs CLN-specified polypeptides for activity, is there not only a suggestive positive feedback loop (Cross; Nasmyth et al.), but also a serious boot-strapping problem? The way to solve this is not clear; it was Bruce Futcher, I think, who suggested that this function might be performed by low levels of CLN3, which does not show the cell-cycle-connected fluctuations of the other two CLN gene products (Tyers et al.). This may or may not be right, but it is a testable hypothesis. We return below to some other problems raised by the CLN genes and their properties. Whether or not the proposed positive feedback loop exists is in many ways beside the important point, which is that the data show that Cdc28 is capable of turning on transcription sharply and requires active CLN gene products to do so. The transcription of a large family of genes involved in DNA synthesis shows a striking increase in transcription at this time in the cell cycle (Johnston et al.). It is very likely that whatever factor binds to and activates the MluI restriction site that marks these genes will prove to be regulated by cyclins and Cdc28. This is an exciting new breaking area in the field, with clear implications for START control in higher eukaryotes. It is clear that at least part of the activation of resting mammalian fibroblasts comprises sequential waves of production of mRNA for leucine zippers, zinc fingers, helix-loop-helixes, and doubtless more to come. But

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before we get carried away with the idea that START is essentially a kind of three-stage booster rocket of transcriptional cascades ~t la bacteriophage A, a note of caution should be sounded. First, this is emphatically not the way the yeast people see it. Lee Hartwell explained carefully to me that probably the concentration of all the known components of the D N A synthesis machinery is sufficient to carry out several rounds of replication and cell division. I cannot see that this is a serious objection, because despite the impressive advances by Ferguson et al., Erdile et al., Dutta et al., Laskey et al., Milarski et al., and Fotedar and Roberts, and despite a steady unearthing of shards of evidence that phosphorylation by cyclin/cdc2 kinases may be necessary, the fact is we do not yet know how replication is initiated on cellular origins, in terms either of the requisite components or of their regulation, i.e., some component whose synthesis is required afresh each S phase may yet be found. It is too early to tell, although it will come. Nevertheless, it is crucial to distinguish between the provision of the essential components of the replication machinery, on the one hand, and the switch that activates replication, on the other. It is practically certain that replication is activated by posttranslational modification of a protein or proteins. The provision of those proteins by transcription and translation is obviously a prerequisite for the switch, and disentangling the details may be difficult. Going back to START, and a question that was asked on the first morning of the meeting: How long is START, and when does it begin and end? Is there, for example, any sense in which it may be apt to consider the beginning of START as equivalent to the onset of M phase, and the end of START as analogous to the metaphase/anaphase transition? In other words, high levels of SPF are found before S phase actually begins, and by the time the activating switch is thrown, SPF is no longer present. This would represent a kind of escapement mechanism. Perhaps these are silly ideas and questions, too vague and hypothetical, but I do not think so. I also think it will not be very long before the people who work on Saccharomyces cerevisiae (many of the same people who worked with bacteriophage A) will provide us with clear answers. Yeast is informative in other ways as well. Yet, despite much work, many beautiful diagrams, and striking analogies with signal transduction pathways (for review, see Marsh et al. 1991), it is not yet known how a-factor stops the cell cycle before START. The FAR1 story from Fred Chang was elegant and simple last year, when the gene product of FAR1 was thought to be a transcriptional repressor. This year, it is no longer a transcriptional repressor (Herskowitz et al.), which makes it much more interesting. Far1 may control the stability or translation of CLN2 mRNA, or the stability of CLN polypeptides, or their ability to activate Cdc28. However it turns out, the details will surely be fascinating and, with any luck, instructive for higher eukaryotes.

Multiple Cyclins Combine with a Family of cdc2-related Kinase Subunits However the CLN genes regulate START in yeast, the triple cln- deletion strain of yeast has provided an excellent way to find new cyclins in higher organisms, and the description of these discoveries formed some of the latest and most exciting news in this volume. We now have cyclins A, B1, B2, C, D1-3, and E from humans, CLN1-3 and CLB1-4 in budding yeast, and pucl (Forsburg and Nurse 1991b), cigl, and cdcl3 in fission yeast. It is unlikely this list is complete. Some of the cyclins (the new-found C family) are somewhat distant relatives, and the regulated destruction that drew attention to cyclins A and B in the first place has yet to be studied in detail for the nonmitotic members of the family. Actually, in early Drosophila embryos, it is still not clear if the mitotic cyclins undergo programmed destruction during the rapid early cycles of the syncytium (Glover et al.), and there is a nagging question lurking of whether such rapid cyclical activation and inactivation of cyclin/cdc2 could perhaps employ some other mechanism of regulation, or even (breathe it not in Gath) involve yet some other way altogether of "driving" the cell cycle. Members of the cyclin D family were discovered at almost exactly the same time in no less than four laboratories, using three quite different kinds of approaches; apart from the triple CLN deletion strain complementation assay (Matsumoto and Beach; Reed et al.), Arnold et al. found it overexpressed in parathyroid tumours, and it seems actually to be the bcl-1 oncogene (Withers et al. 1991). Sherr's laboratory (Matsushime et al.) came upon cyclin D by a standard search (superbly executed, it must be said) for genes expressed in macrophages induced to proliferate by the peptide growth factor CSF-1. This is perhaps the most promising sign that there is a connection between cell signaling molecules and the cell cycle engine, although there is no evidence yet that cyclin D is either necessary or sufficient for cell growth and division (Matsushime et al.; Arnold et al.). The evidence is still circumstantial, and since every single cyclin known so far seems to be able to complement the CLN genes (Reed et al.; Fotedar and Roberts), a warning note should sound. It appears as though any generic cyclin can somehow rescue this strain of yeast, as we discussed previously when thinking about the targeting functions of cyclins. Why are there so many cyclins, and what do they all do? Equally, what is the meaning of the discovery of several new members of the cdc2 family of proteins, or cdk family as was decided here to call them (cdk can stand for cell division kinase or cyclin-dependent kinase, and in the long run, Cdc28 and cdc2 should be renamed cdkl) (Meyerson et al.). It remains to be seen whether some of the wilder members of the family of homologs, like P C T A I R E or PSSALRE, do in fact require companion subunits, or indeed whether they have anything to do with cell cycle control whatsoever.

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SUMMARY But we need names to call things by, and an open meeting of interested parties decided on balance that cdk2 (3, 4, etc.) is better than cdc2B, cdc2C, cdc2D, etc., there being ony 26 letters in the alphabet. The field would do well to give some attention to nomenclature, which is exceedingly confusing. At the very least, a rationalization should be attempted between the two kinds of yeast and Aspergillus. To call two completely different genes by the same name, cdc25, is terrible for everyone and to call the same gene by different names, like cdc2 and CDC28, is certainly not helpful for newcomers. The kinase subunits cdc2 and cdk2 (previously known as Egl, because it was found in Xenopus eggs by Paris et al. 1991) can each associate with cyclin A (Meyerson et al.; Kobayashi et al.). So far, there is clear evidence for binding of cyclins B1 and B2 to p34 cdc2, whereas the association of these two mitotic cyclins with cdk2 is not proven. Although cyclin D appears to be associated with a polypeptide that can be recognized by anti-PSTAIRE antibodies, this is probably neither cdc2 nor cdk2 (Matsushime et al.). There is excellent evidence that cdc2 is required for both S T A R T and mitosis, but evidence was newly presented at the meeting, and subsequently published by Fang and Newport (1991), that cdk2 is necessary for D N A synthesis in Xenopus embryos, confirming and extending the older report from Blow and Nurse (1990). It is not clear why cdk2, and not apparently cdc2, can do this. The companion subunit of cdk2 for this function is still not identified (Mailer et al.). When it comes to the newer members of the cyclin and cdc2 family, it is too soon to offer any opinion. It will probably be hard to discover the precise functions of all these protein kinases, even if they have yeast counterparts. A simple diagram of the present state of cyclin-cdk interactions, with no attempt at guessing function, is shown in Figure 3. It is unfortunate that we still do not know how the original cyclins A, B1, and B2 really differ from one another. Everyone is agreed, apart perhaps from peo-

cyclin family Missing kinase partners

I

? - - cyclinE ? -- cyclin D

/

cyclin B2

cdc2 -- cyclin B1

\ /

cdk2

r

A

\?

PCTNRE-

?

Missing cyclin partners

PITAIRE- ?

cdc2

family

Figure 3. The cyclin and cdk family.

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pie who work on Drosophila, that cyclin A operates in the nucleus, and cyclin B1 is in the cytoplasm (Pines and Hunter; Glover et al.; Lehner et al.). In Drosophila, both cyclins are bound to microtubuleassociated proteins and thence to microtubules (Kellogg et al.), but here the agreement ends. Cyclin A has been implicated in S phase (Fotedar and Roberts; Dutta et al.) and in M phase (Lehner et al.), and Walker and Maller (1991) recently proposed that cyclin A controlled the D N A synthesis completion checkpoint. Finally, both Dor6e and Karsenti (Devault et al. ; Buendia et al.) suggest almost exactly the opposite, providing evidence that cyclin A/cdc2 kinase may be required to activate cyclin B/cdc2 kinase. Can they all be right? I do not know, but it is hard to believe. To further confound the issue, cyclin A turns up in ever more surprising company, binding to adenovirus E1A protein, the retinoblastoma and p107 gene family, and perhaps most surprisingly, the transcription factor E2F (Meyerson et al.; Nevins et al.). The significance of these interactions remains to be seen, but they tend to reinforce the idea that cyclins help determine the substrate specificity of their companion kinase subunit, besides promoting transport to certain subcellular compartments and playing some role in the timing of activation of the kinase. It is not even quite clear whether cyclins are invariably associated with a protein kinase subunit or whether they can affect the activity of other kinds of proteins besides the cdc2 family, perhaps by acting as subcellular targeting subunits. If this should prove to be true, it raises an alarming specter of unfathomable multiple interactions. As Brenner (1981) puts it: "The binding site is the heart of the matter; unlike a computer where a signal is sent from one part to another by a wire connecting the two, [the cell] does not have physical addresses and uses a broadcast system instead with logical addresses: each [enzyme] simply ignores the irrelevant messages and only those fitting its binding site are retained and acted upon. It is this special hardware that enables [the cell] to be an effective multiple parallel system without paying an enormous tax in elaborate wiring diagrams. Similar arguments apply to the control systems, and it is easy to show that regulation works on the basis of locally controlled anarchic demons bound together only by a global property of resource partitioning in which greed is punished by death. There is no master control, a single supervisor or monitor, as some might once have assumed, based on intuitive experience of man-made systems." (Brenner's italics.) I have always liked this passage, and it seems as applicable to cell cycle control in 1991 as it was to questions of development in 1981. If not more so. All the same, we welcome the new members of both the cdc2 and cyclin families, anticipating that it may be some time before we find out what they all do. As everyone knows (except in my teaching experience, the optimistic young, who often evince a touching faith in computer modeling, prediction, and other mystical be-

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liefs), this is a very serious difficulty, so serious that in our innermost hearts, we know that it is not really the best way to do biology. I was once the guardian of a medieval device of long-forgotten function known in Cambridge as The Butter Measure. It probably was for measuring corn, not butter, and it was by no means obvious how it could be used to measure anything at all. The point is, although things can be designed for a particular function, or evolved to fulfill a particular function, function can never be deduced from structure without other information, usually shared features with familiar objects. Although the null phenotype of a gene often gives a clue, often it does not. When more modern technology is brought to bear, in the form of antisense D N A and R N A or antibodies, one needs lots of controls and extremely cautious interpretation to be sure of any positive conclusions. Much to be preferred, and almost always telling, is the organic chemist's approach of reconstruction, now made possible by a variety of excellent cell-free systems that can perform complex parts of cellular functions in accessible form. Quite the most striking example of this at the meeting was John Kilmartin's artificial alteration of the length of the yeast spindle pole body (SPB). He identified a gene that encoded one of the structural components of this organelle (Rout and Kilmartin) and made a prediction based on protein homologies, tested it by using sitedirected mutagenesis to alter the length of the protein, and confirmed his expectation by electron microscopy. Shortening the protein brings the layers of the SPB closer together, lengthening it has the opposite effect. In fact, the field of the cell cycle has made much use already of this kind of approach, principally in the use of very crude cell-free systems lacking particular components or oversupplemented with others. For a geneticist's view of the spindle pole and the complexities of interpreting phenotypes, see the excellent short review by Winey et al. Centrosomes and kinetochores are among the most mysterious organelles at present, particularly the formed. It is reasonably easy to imagine how kinetochore duplication might be achieved if they are built on a core of D N A (for an approach to defining kinetochore components, see Earnshaw et al.). It is by no means clear how centrosome replication is limited to one copy per cell cycle, and both Breck Byers (Winey et al.) and John Kilmartin (Rout and Kilmartin) are firmly committed to this problem, which is most likely to yield first in yeast. The consequences of not having two centrosomes are lethal, as Boveri (1902) realized almost a century ago. The function of centrosomes and kinetochores is not surprisingly controlled by reversible phosphorylation, and beautiful new optical methods as well as clever chemistry are being brought to bear on these very difficult architectural problems (Buendia et al.; Hyman and Mitchison).

Recessive Oncogenes and Cell Cycle Control Eggs is eggs: What can one say about how "real" cell cycles are controlled? Rec~nt successes with yeast gen-

etics and analysis of embryonic cell cycles have yet to greatly deepen understanding the control of the G1/S transition in mammalian cells. It is perhaps significant that with the exception of PRAD1/bcll/cyclin D, oncogenes have not provided a rich haul of genes that comprise the heart of the cell cycle engine. On the other hand, there are certainly some suspicious coincidences when it comes to considering the retinoblastoma oncogene, and the ways in which adenovirus, human papillomavirus type 16 (HPV-16), and SV40 can transform cells. I shall deliberately ignore p53 and direct readers to Zambetti et al. and Prives et al. for enlightenment on what I find too confusing a subject to attempt any kind of exegesis. Moreover, the connections between p53 and other cell cycle regulatory genes at present seem flimsy, whereas connections between cellular retinoblastoma (RB) and p107 proteins, and adenovirus E1A, SV40 large T antigen, and HPV16 E7 proteins with each other and the cell cycle control machinery, are almost embarrassingly rich. Everything binds everything, and at the moment, a particular transcription factor, known as E2F, seems to hold center stage. E2F was originally isolated as a cellular protein that was required for transcription of the adenovirus E2 gene. It is a DNA-binding protein that is usually detected by D N A bandshift assays. The D N A recognition sequence is well defined, and rather like the MluI factor in yeast, the E2F recognition motif turns up in a number of genes that show S-phase connections (Nevins et al.). It turns out that E2F is found in association with RB, and also with cyclin A. It is too soon to know if these findings represent a great truth, a snark, or a boojum. I think I understand and can explain what people would like to think, which may be useful, because the underlying ideas are not always explicitly stated in the primary literature. The first fact to realize is that RB is a relatively abundant nuclear protein (Chittenden et al.). The second fact is that it undergoes changes in its phosphorylation state through the cell cycles (Mittnacht et al.). Third, it contains several potential sites for cdc2 kinase and can indeed be phosphorylated by purified cdc2 kinase in vitro (Lees et al. 1991). Finally, RB immunoprecipitates contain cyclin A and vice versa, and the same is true of p107, which is a related protein that shows some degree of sequence similarity in the regions of these proteins that appear to be responsible for binding other proteins, which include adenovirus E1A, SV40 large T antigen, and HPV-16 E7. The general idea is very simple. It is thought that proteins like RB and p107 normally bind transcription factors like E2F and c-myc and inhibit their activity. Turning on promoters that need E2F or c-myc is seen as simply a matter of squeezing the transcription factors out of these sponge-like repressors, or as Lee et al. put it, corrals. There are two ways of doing this. One, the natural way, is to phosphorylate RB, using an appropriate cyclin/cdk combination. The other way, which is used by transforming viruses, which may need to activate the cellular S-phase machinery in order to support their own replication, is to compete

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SUMMARY with the transcription factors for binding to the RB "pocket." So, adenovirus E1A would act by flushing out RB and releasing E2F, which would then activate the transcription of genes that are necessary for S phase. In normal use, the cycle is reset after mitosis, when RB normally reverts to its underphosphorylated state in which it can once more sop up all the E2F. Whether or not this is strictly correct remains to be seen, but at least it provides a simple conceptual model, which has the virtue of integrating a number of otherwise seemingly disjointed and hard-to-remember (at least for the outsider) facts. I hope this is not a complete travesty of what is intended by Drs. Harlow, Lee, Livingston, and Weinberg (Meyerson et al.; Lee et al.; Chittenden et al. ; Mittnacht et al.). What was particularly striking about the approaches of these groups was the widespread and imaginative use of the new protein chemistry that clone-sequence-and-express technology provides, together with affinity chromatography of various ingenious kinds to reconstruct and probe protein:protein interactions in cells and test tubes.

Dominant Oncogenes and Cell Cycle Control According to the checkpoint idea, most cell cycle control genes will be recessive to their wild-type counterparts. How then do dominant oncogenes fit into the scheme of things? It is not hard to understand how dominant alleles of ras work; they are activated by mutation and continue to give an inappropriate signal long after the wild-type protein's signal would have decayed. The only problem is in knowing what process(es) ras normally monitors, and which way the logic works. Several of the known oncogenes are protein kinases, but not many of these were discussed at the Cell Cycle meeting. An idea of how tyrosine kinases transform cells was clearly set out by Williams et al., who introduced me to the idea that tyrosine phosphate was used as a flag on receptors to announce occupancy and that the setting of the flag was read, as it were, by specific protein:protein interactions. The cytoplasmic tail of the P D G F receptor, it seems, is a patchwork of signaling domains that can bind other effector molecules, like phospholipases, inositol kinases, and perhaps G proteins. Evidently, tyrosine phosphate can make a strong noncovalent bond with another protein, and the particular context of a given phosphorylatable tyrosine can provide a highly specific dock for particular proteins. When they bind together, they constitute a little protein machine that propagates the external signal. There is an important distinction between this kind of idea and the original protein kinase paradigm, by which phosphorylation altered the activity of an enzyme. Not that the two are mututally exclusive; it is still not clear, for example, whether binding phospholipase C to the phosphorylated P D G F receptor is enough to activate it or whether having bound, the phospholipase then undergoes covalent modification, which turns it on. Serine/threonine kinases, like raf (Mamon et al.)

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and mos (Daar et al.), are perhaps easier to understand in principle, but more mysterious in practice. They seem to operate in the cytoplasm, but their normal functions and abnormal substrates are still largely obscure, mos is currently the more telling and has a closer connection with the cell cycle, considering its role in oocyte maturation and the fertilization checkpoint. It is still not known how mos contributes to the stabilization of MPF activity in mature X e n o p u s eggs, except that it is probably bound to microtubule-containing struct u r e s - t h e targeting leitmotif again (Daar et al.). It was always puzzling, however, whether rnos tended to cause M-phase arrest and how it could act as a transforming gene, the exact opposite of cell cycle arrest. A potential answer to this paradox comes from consideration of another activity that mos possesses, its ability to induce oocyte maturation. The transition from G 2 to M phase in an oocyte represents something rather more than that, because the oocytes have been arrested in G 2 or meiotic prophase for so long. They are quiescent cells awaiting a hormonal signal for activation, and mos is on the signaling pathway. Thus, artificial oocyte activation by microinjection of translatable m R N A or active mos kinase can bypass the normal control. Unfortunately, there is no term to describe the equivalent of G O when it comes to G e cell cycle arrest, but I suggest that if mos can break the G 2 arrest of oocytes, it probably does much the same for G Oarrest in normal cells. It is probably this ability, rather than the CSF connection (which is not yet fully understood), that accounts for the potency of c-mos and v - m o s as oncogenes. Finally, although transcription factors like myc and l o s present few conceptual difficulties, particulars of how they promote cell cycle progression--how close they are to the control system of the cell cycle engine-are still suprisingly sparse, considering the tremendous wealth of details available. It is certainly encouraging that regulated misexpression of m y c can prevent cells from stopping in G O and start G O cells growing again (Bishop et al.). An interesting point, which Bruce A1berts (pers. comm.) raised in a question, was whether this progression should be ascribed to the activation of transcription from the presumptive m y c - d e p e n d e n t promoters or rather perhaps of flushing out the RB pockets, since it seems that m y c can be found in association with RB (Rustgi et al.; Lee et al.; Chittenden et al.). In other words, transformation by myc may be like transformation by E1A and works by turning on E2F or some such transcription factor, and not necessarily as a result of its own transcriptional activity. This is a significantly different way of looking at the picture of multiple protein:protein interactions that crop up again and again in transcription factors (Bishop et al.; Blackwood et al.; McKnight, pers. comm.). Meanwhile, the study of periodic transcription coupled to the cell cycle is in its infancy, and it does not take a crystal ball to see that this will soon change and that reversible phosphorylation will play a crucial part in the regulation (Segil et al.).

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CONCLUSION To summarize an intense week of concentrated data has not been easy, and I apologize for my selective memory, which tends to retain only what fits my preconceived n o t i o n s - - a n d obsessions. I listened to every single talk and heard not one dull or bad one among the scores that were presented. Likewise, almost every poster, and there were hundreds of them, contained something of interest, alas not reflected in this volume. If their efforts have gone unrecognized, ascribe it to the problem of time and space, and just keep trying to explain. The penny will eventually drop. I have said nothing about the regulation of bacterial cell cycles, which were elegantly introduced by Rothfield et al. and Trun et al. and which have sharp lessons for higher eukaryotes. The details of mitosis were not as thoroughly ignored as they have been here, as the table of contents reveals. When I was worrying about what to say and write in summary of the diverse strands that comprise studies of the cell cycle, I wondered whether it was possible to find an apt metaphor that would describe the assembly of protein machines into working factories, and perhaps simplify the task of classifying and encompassing all these disparate thoughts and systems. As I walked around the Cold Spring Harbor campus, and poked my nose into the new buildings, still being fitted out, the architectural analogy kept on recommending itself. Unfortunately, I wasn't trained as an architect. My apologies to everyone whose ideas I have either ignored or reproduced without attribution.

REFERENCES

Bischoff, F.R. and H. Ponstingl. 1991. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature 354: 80. Blow, J.J. and R.A. Laskey. 1988. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332: 546. Blow, J.J. and P. Nurse. 1990. A cdc2-1ikeprotein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell 62" 855. Bourne, H.R. 1988. Summary: Past, present, and future. Cold Spring Harbor Symp. Quant. Biol. 53: 1019. Boveri, T. 1902 (1964 translation). On multipolar mitosis as a means of analysis of the cell nucleus. In Foundations of experimental embryology (ed. B.H. Willier and J.M. Oppenheimer), p. 74. Prentice-Hall, Englewood Cliffs, New Jersey. Brenner, S. 1981. Genes and development. In Cellular controls in differentiation (ed. C.A. Lloyd and D.A. Rees), p. 3. Academic Press, London. Carr, D.W., R.E. Stofko-Hahn, I.D.C. Fraser, S.M. Bishop, T.S. Acott, R.G. Brennan, and J.D. Scott. 1991. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J. Biol. Chem. 266: 14188. Curtis, H. 1983. Biology, 4th edition. Worth, New York. Ducommun, B., P. Brambilla, M.-A. F61ix, B.R. Franza, Jr., E. Karsenti, and G. Draetta. 1991. cdc2 phosphorylation is required for its interaction with cyclin. EMBO J. 10: 3311.

Fang, F. and J.W. Newport. 1991. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell 66: 731. Ferrell, J.E.J., M. Wu, J.C. Gerhart, and G.S. Martin. 1991. Cell cycle tyrosine phosphorylation of p34cat2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs. Mol. Cell. Biol. 11: 1965. Finlay, D. and V. Chau. 1991. Ubiquitination. Annu. Rev. Cell Biol. 7: 25. Forsburg, S.L. and P. Nurse. 1991a. Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Annu. Rev. Cell Biol. 7: 227. --. 1991b. Identification of a Gl-type cyclin pucl § in the fission yeast Schizosaccharomyces pombe. Nature 351: 245. Glotzer, M., A.W. Murray, and M.W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349: 132. Gotoh, Y., K. Moriyama, S. Matsuda, E. Okomura, T. Kishimoto, H. Kawasaki, K. Suzuki, I. Yahara, H. Sakai, and E. Nishida. 1991. Xenopus M phase MAP kinase: Isolation of its cDNA and activation by MPF. EMBO J. 10: 2661. Gould, K.L., S. Moreno, D.J. Owen, S. Sazer, and P. Nurse. 1991. Phosphorylation at Thr 167 is required for Schizosaccharomyces pombe p34cat2 function. EMBO J. 10: 3297. Hartwell, L.H. and T.A. Weinert. 1989. Checkpoints: Controls that ensure the order of cell cycle events. Science 246: 629. Hartwell, L.H., J. Culotti, J.R. Pringle, and B.J. Reid. 1974. Genetic control of cell division cycle in yeast. Science 183: 45. Kinsella, B.T. and W.A. Maltese. 1991. Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2. Nature 350: 715. Krek, W. and E.A. Nigg. 1991. Mutations of p34cat2 phosphorylation sites induce premature mitotic events in HeLa cells: Evidence for a double block to p34cdc2kinase activation in vertebrates. E M B O J. 10: 3331. Lees, J.A., K.J. Buchkovitch, D. Marshak, C.W. Anderson, and E. Harlow. 1991. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 10: 4279. Lundgren, K., N. Walworth, R. Booher, M. Dembski, M. Kirschner, and D. Beach. 1991. mikl and weel cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64: 1111. Marsh, L., A.M. Neiman, and I. Herskowitz. 1991. Signal transduction during pheromone response in yeast. Annu. Rev. Cell Biol. 7: 699. Masui, Y. and C.L. Markert. 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177: 129. Mazia, D. 1959. Cell division. Harvey Lect. 53: 130. --. 1961. Mitosis and the physiology of cell division. In The cell (ed. J. Brachet and A.E. Mirsky), vol. 3, p. 77. Academic Press, New York. Mitchison, J.M. 1970. The biology of the cell cycle. Cambridge University Press, Cambridge, United Kingdom. Murray, A.W. and M.W. Kirschner. 1989. Dominoes and clocks: The union of two views of the cell cycle. Science 246: 614. Norbury, C., J. Blow, and P. Nurse. 1991. Regulatory phosphorylation of the p34cat2 protein kinase in vertebrates. E M B O J. 10: 3321. Osmani, A.H., K. O'Donnell, R.T. Pu, and S.A. Osmani. 1991. Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint. EMBO J. 10" 2669. Paris, J., R. Le GueUec, A. Couturier, K. Le Guellec, F. Omilli, J. Camonis, S. MacNeill, and M. Philippe. 1991. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Natl. Acad. Sci. 88: 1039. Payne, D., A. Rossomando, P. Martino, A. Erickson, J. Her,

Downloaded from symposium.cshlp.org on July 6, 2016 - Published by Cold Spring Harbor Laboratory Press

SUMMARY J. Shabanowitz, D. Hunt, M. Weber, and T. Sturgill. 1991. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). E M B O J. 10: 885. Rao, P.N. and R.T. Johnson. 1970. Mammalian cell fusion studies on the regulation of DNA synthesis and mitosis. Nature 225: 159. Rapkine, L. 1931. Sur les processus chimiques au cours de la division cellulaire. Ann. Physol. Phisicochim. Biol. 7: 382. Sola, M.M., T. Langan, and P. Cohen. 1991. p34 cdc2 phosphorylation sites in histone HI are dephosphorylated by protein phosphatase 2A1. Biochim. Biophys. Acta 1094: 211. Surana, U., H. Robitsch, C. Price, T. Schuster, I. Fitch, A.B. Futcher, and K. Nasmyth. 1991. The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae. Cell 65: 145. Tuomikoski, T., M.-A. Felix, M. Dor6e, and J. Gruenberg.

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1989. Inhibition of endocytic vesicle fusion by the cell cycle control protein kinase cdc2. Nature 342: 942. Walker, D.H. and J.L. Mailer. 1991. Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 354: 314. Withers, D.A., R.C. Harvey, J.B. Faust, O. Melnyk, K. Carey, and T.C. Meeker. 1991. Characterization of a candidate bcl-1 gene. Mot. Celt. Biol. 11: 4846. Wu, M. and J.C. Gerhart. 1980. Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis. Dev. Biol. 79: 465.

This list is not intended to be comprehensive. Apart from a sprinkling of classical articles and reviews where I have quoted verbatim, citations in this list are to articles that appeared after the Symposiumor cover points that were not addressed at the meeting. I hope this may be useful.

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Summary: Put Out More Flags T. Hunt Cold Spring Harb Symp Quant Biol 1991 56: 757-769 Access the most recent version at doi:10.1101/SQB.1991.056.01.085

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