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Cell multiplication Editorial overview Steven I Reed* and James L Mallert Addresses *The Scripps Research Institute, Department of Molecular Biology, MB-7, 10550 N Torrey Pines Road, La .Iolla, CA 9203?, USA; e-mail: [email protected] tHoward Hughes Medical Institute and Department of Pharmacology, University of Colorado, School of Medicine, Denver, CO 80962, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:763-766 © Current Biology Ltd ISSN 0955-0674 Abbreviations CAK CDK-activating kinase CDK cyclin-dependent kinase pRb retinoblastorna protein

T h e placement of cyclin-dependent kinases (CDKs) at the center of all hierarchies of cell cycle control is undeniably the overarching theme of the decade in the fields of biology that bear directly on cell growth and proliferation. Whereas initially the emphasis was on cataloging the various components and regulatory motifs of CDK systems in yeasts and metazoans, in recent years work has shifted to establishing actual mechanisms whereby signaling pathways interface with these CDK systems to effect control of proliferation. T h e results have been decidedly mixed. Some of the simple models appear to have survived the test of serious experimental scrutiny, but others have been perplexingly difficult to either validate or exclude. In particular, mice rendered nullizygous for elements that either regulate CDKs or are regulated by them have exhibited phenotypes that both support and contradict current dogma. Another area where progress has been offset by uncertainty is the role of CDK phosphorylation in biological control of the cell cycle. Even though mechanisms of regulatory phosphorylation of CDKs have been well established at the biochemical level for years, their contribution to CDK regulation in the context of cell cycle control remains unsettled (see below). T h e contributions in this section of Current Opinion in Cell Biolo~ provide a balance between recent unambiguous conceptual advances and the ambiguities that stubbornly persist, hopefully indicating where future research efforts need to be redoubled. Arguably, the most unequivocal success story in the cell cycle arena has been the ongoing structural analysis of CDKs in complex with impo.rtant regulatory proteins (Morgan, pp 767-772). Building upon their initial crystallographic determination of the structure of human cyclin A bound to human CDK2, Pavletich and colleagues have solved the structures of both the cyclin A-CDK2 complex that displays an essential positive regulatory

phosphorylation, and the cyclin A-CDK2 complex that is bound to an active fragment of the inhibitor p27Kipl. T h e former structure reveals how activation of CDKs is accomplished as a two-step process, with cyclin binding and phosphorylation of the so-called T-loop each contributing to reconfiguration of the CDK active site. T h e structure of the p27Kipl-cyclin A-CDK2 ternary complex, however, is by far the most dazzling and provocative. Pavletich and colleagues showed that the amino terminus of the inhibitor anchors to a conserved groove in the cyclin fold, allowing the molecule to stretch across the amino-terminal lobe of the kinase and invade its active site. A tyrosine of the inhibitor molecule inserts into the purine-binding pocket of the kinase, precluding the binding of ATP. Not only does this structure provide a rationale for some of the numerous biochemical observations that have preceded it, for example, the demonstrated affinity of p27 and structurally related (so-called Cip/Kip) inhibitors for both cyclins and CDKs, but it also enters the fray of the stoichiometry controversy that continues to be associated with these inhibitors. Although it has been claimed that a stoichiometry of more than one Cip/Kip inhibitor molecule per C D K complex is required to inhibit a C D K complex, the structure clearly favors the idea that one molecule should be sufficient, some caveats notwithstanding. Details of the second important CDK complex structure to emerge in the past year are those of CDK2 bound to CksHsl (Morgan, pp 767-772). T h e latter protein represents a family of small, evolutionarily conserved proteins (of which the archetype is the fission yeast protein sucl) that, for more than ten years, have been known to interact genetically with CDKs and to physically associate with them. However, knowledge of the function(s) of Cks proteins has remained elusive. T h e structure presented by Tainer and colleagues indicates that Cks proteins bind to a conserved region of the carboxy-terminal lobe of CDKs, far from the active site and from domains known to regulate activity. Furthermore, Cks binding does not alter the structure of CDK, significantly ruling out allosteric regulation of kinase activity. These findings lead to the conclusion that Cks proteins must serve as adapter molecules that allow CDKs to interact with important targets or regulators. Biochemical and genetic studies will clearly have to determine what these targets or regulators might be. Another context in which structure (albeit primary structure) has provided insight into function is the analysis of checkpoint controls. In both the checkpoint that monitors spindle integrity (Rudner and Murray, pp 773-780) and that which monitors genome integrity (Stewart and

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noch, pp 781-787), signaling elements that provide )mmunication between the cell's internal environment ad the machinery of cell cycle progression are structurally reserved from yeast to man. T h e finding that Mad2, kinetochore-associated protein, originally identified in ~ast, has a structural homolog in vertebrates suggests that 1 eukaryotes use the same means to couple cell cycle rogression to successful assembly of the mitotic apparatus ~udner and Murray, pp 773-780). T h e MAD2 gene was btained via a screen for budding yeast (Saccharomyces 'revisiae) mutants defective in cell cycle arrest in the ~ntext of activation of the spindle assembly checkpoint. lthough vertebrate homologs of other yeast gene prodcts identified in such screens remain to be found, a mctional model based on both yeast and mammalian 'ork is emerging. In this model, phosphorylation of inetochore-associated proteins in the absence of spindle Etachment generates a signal that blocks progression lto anaphase. Consistent with this hypothesis, it has een possible to remove all chromosomes (and therefore inetochores) from a mitotic spindle by micromanipulation 'ithout blocking anaphase movements of the spindle and absequent cytokinesis. .s with the spindle integrity checkpoint, the checkoints that confer dependence of mitosis upon genome :plication and genome integrity utilize evolutionarily onserved elements (Stewart and Enoch, pp 781-787). If particular interest is the S. cerevisiae MEC1 gene ~hich encodes a large protein that shares homology with hosphatidylinositol-3 (PI-3) kinases. Mutations in MEC1 onfer defects in both the replication and the DNAamage checkpoints. Mutations in a homologous fission east gene, rad3 ÷, confer similar phenotypes. Most jrprisingly, however, mutations in a homologous human ene (ATM, or Ataxia Telangiectasia Mutated) confer syndrome known as Ataxia Telangiectasia, which is haracterized by multiple checkpoint defects associated 6th genome integrity. Thus, once again, signaling pathrays responsible for relaying information about the cell's aternal milieu to the machinery of cell cycle progression re found to be functionally and structurally conserved. L novel twist in this story is the homology among /Iecl, Rad3, ATM and another protein with lipid kinase omology known as DNA-PK (DNA-dependent protein inase). Although DNA-PK shares homology with PI-3 inases, it is in fact a serine/threonine protein kinase Ath no detectable lipid kinase activity. Furthermore, nd most intriguingly, it is activated by double-strand reaks in DNA, thus possibly serving as a paradigm for he homologous DNA checkpoint kinases. Although the trgets (be they protein or lipid) of Mecl and related inases are not known, the S. cerevisiae protein kinase lad53 is an excellent candidate for such a target in hat genetic studies place it downstream of Mecl, it is ubjected to Mecl-dependent phosphorylation in response DNA damage or a DNA-synthesis blockage, and it is ctivated by phosphorylation.

Although conservation of structure and function in checkpoint control is an emergent theme, the converse also appears to be true (Stewart and Enoch, pp 781-787). From a comparison of DNA-checkpoint genes from budding yeast and fission yeast, it is known that a significant number of fission yeast genes have no homologs in budding yeast. This can be stated with confidence as the entire budding yeast genome has been sequenced. Thus, it can be expected that, when checkpoint pathways of vertebrates are elucidated in greater detail, there will be both conserved and distinctive features relative to the yeast checkpoint pathways. Another phylogenetically conserved motif is the regulation of CDKs themselves by phosphorylation. All eukaryotes studied engage in positive and negative regulatory phosphorylations of CDKs. Two reviews in this section deal with this issue from different perspectives (Sclafani, pp 788-794; Lew and Kornbluth, pp 795-804). It has long been established that CDKs require a phosphorylation event on the centrally located T-loop for activation. Yet the enzyme that performs this phosphorylation in vivo (known generically as CAK for CDK-activating kinase) remains a subject of controversy (Sclafani, pp 788-794). Initially, the CAK enzyme, purified as the principal CDK-activating kinase that could be assayed in marine invertebrate and Xenopus egg extracts and mammalian cell extracts, was found to be a CDK-cyclin complex itself, CDK7-cyclin H. Two discoveries, however, have undermined the certainty of this view of CDK activation. First, CDK7-cyclin H was shown to be a component of T F I I H , part of the basal transcriptional machinery, where it apparently phosphorylates the C T D (carboxy-terminal domain) of RNA polymerase II, raising the question of why a single enzyme should regulate such diverse cellular processes. Second, CDK7-cyclin H in S. cerevisiae was shown only to be a component of T F I I H , having an in vivo role in RNA polymerase II dependent transcription, but none in CDK activation. Furthermore, a novel protein kinase, Cakl/Civl, has been identified as the CDK-activating enzyme of S. cerevisiae. In the absence, therefore, of any compelling in vivo evidence that CDK7-cyclin H is the functional CAK of invertebrates and vertebrates, or of the discovery of a Cakl/Civl homolog in organisms other than S. cerevisiae, the issue must remain unresolved. Another unresolved question is that of the role of negative phosphorylation of CDKs in cell cycle control (Lew and Kornbluth, pp 795-804). In particular, the phosphorylation of a tyrosine residue (usually YI5, for example in CDK2) near the amino terminus of most CDKs has been identified as an important regulatory event in the context of checkpoint controls. T h e enzymes that phosphorylate and dephosphorylate Y15 are known. There is, however, little consensus on how the phosphorylation state of CDKs is actually regulated when these checkpoints are activated. To add to the confusion, mutations of Y15 to F (Phe; or equivalent mutations) appear to have different impacts on

Editorial overview Reed and Mailer

checkpoint control in different organisms. For example, the Y15F mutation appears to completely abrogate or severely impair the DNA-synthesis and DNA-damage checkpoints in S. pombe but has no effect on these checkpoints in S. cerevisiae. T h e effects in other organisms fall somewhere in between these extremes. Lew and Kornbluth (pp 795-804) suggest two possible models to explain these differences. In the first model, two mechanisms, one dependent on Y15 phosphorylation and one independent of this, coexist in all organisms, with the prevalence of one mechanism over the other in any cell type, accounting for the observed differences. T h e second model assumes that checkpoint-mediated cell cycle arrest is based not on activating Y15 phosphorylation, but on generating a distinct, as yet uncharacterized, inhibitory signal. This Y15-independent signal, however, needs its amplitude adjusted to the background level of Y15 phosphorylation in the cell in order to be effective. In this model, S. cerevisiae, which executes DNA-checkpoint controls in the context of a cell cycle with low background levels of Y19 phosphorylation (the equivalent of Y15 phosphorylation in other CDKs), would utilize a strong Y19-independent checkpoint signal such that the Y19F mutation would be expected to have little effect. S. pombe, on the other hand, which exhibits a high background level of Y15 phosphorylation, would require this dampening of CDK activity in order for checkpoint signals to achieve cell cycle arrest and, therefore, the Y15F mutation would have a profound effect. Whichever model is correct, the Y15-independent mechanism of cell cycle arrest has remained elusive, although some experiments have suggested that a titratable CDK inhibitor may be involved. One of the checkpoints for which a molecular basis is finally emerging is the so-called restriction point of mammalian cells (Bartek, Bartkova and Lukas, pp 805-814). Bartek, Bartkova and Lukas discuss the journey from the initial phenomenological description of the restriction point by Pardee [1] and colleagues to the present day world of nullizygous mice. At this point, it appears that the central regulatory component accounting for most of the restriction-point phenomenology is the tetinoblastoma protein, pRb. T h e model best supported by currently available data is that pRb exerts negative control on the Gi--+S-phase transition by sequestering positive regulators of growth and cell cycle progression, such as transcription factors of the E2F family. T h e restriction point is passed upon phosphorylation of pRb by CDKs, resulting in the release of the positive effectors. That said, it is not nearly so clear how the other elements invoked in Pardee's model of 20 years ago [1] figure in the modern world of CDKs and their regulators. For example, the unstable restriction-point protein predicted by Pardee and colleagues on the basis of studies with inhibitots of protein synthesis may not exist. Instead, a more complex interplay between cyclins and CDK inhibitors may account for the restriction-point phenomenology. Furthermore, although cyclins, C D K inhibitors and pRb

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itself are clearly part of the restriction-point system, experiments with nullizygous mice have been sobering reminders that even complex models can be overly simplistic. Although p27Kipl is thought to be an important regulator of CDKs involved in pRb phosphorylation, and p27-nullizygous mice show some defects attributable to aberrant growth control, fibroblasts obtained from these animals show a normal response to serum starvation, one of the hallmarks of restriction-point control. Fibroblasts obtained from pRb-nullizygous mice, although exhibiting an attenuated restriction-point control, have not lost the ability to respond to serum deprivation, indicating that pRb-independent mechanisms must account for growth factor regulation of the restriction point. In contrast, fibroblasts from pRb-nullizygous mice have apparently lost their sensitivity to inhibitors of protein synthesis, consistent with the idea that unstable proteins might regulate pRb within the context of restriction-point control, perhaps accounting for the original Pardee results. One additional issue that remains to be resolved is the contribution of dysfunction of the restriction point regulatory system to human malignancy. T h e past few years have seen an abundance of evidence that mutations in elements that have a direct impact on the regulation of pRb, including pRb itself, are frequently associated with cancer. Yet it is not clear whether it is loss of restriction-point control that leads to malignancy or whether deregulation of other functions of the same proteins is responsible for malignant transformation. Although the restriction point, in its purest pRb-dependent sense, is a checkpoint that regulates cell cycle commitment and entry into S phase only in higher eukaryotic cells, the mechanism by which cells actually initiate DNA replication appears to be evolutionarily conserved from yeast to man (Chevalier and Blow, pp 815-821). In the past year, homologs of proteins involved in various aspects of initiation of DNA replication in yeast have been found in frogs and humans. These include the ORC (origin recognition complex) proteins, which presumably establish a chromosomal target for initiation, and the MCM (minichromosome maintenance) proteins, which control the teplicative competence of origins. Analysis of the behavior of MCM proteins in replication-competent frog egg extracts suggests that they are likely to constitute the 'licensing factor' described years ago operationally as the means of periodically regulating origin competence so that only one cycle of replication occurs per cell division cycle. MCM protein complexes appear to bind chromatin in a manner inversely related to their phosphorylation state, providing a possible clue as to how origin licensing may be coupled to the cell cycle program. However, the events that actually trigger origin firing during S phase still remain elusive. We end this overview with a short discussion of the role of proteolysis in cell cycle regulation. Although not a major topic in the current section of Current Opinion in Cell

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Biology, an article was devoted to it in last year's volume [2]. Ubiquitination and degradation of proteins appear to be essential at several crucial cell cycle transitions. This is best understood in yeast, where a specific CDK inhibitor, Sicl, must be ubiquitinated and degraded at the end of G1 phase in order to initiate S phase. Many of the components that are responsible for this process have now been identified, some in the past year, although their precise molecular roles remain to be established. At the other side of the cell cycle, a different set of regulators and ubiquitination targets control both the transition from metaphase to anaphase and exit from anaphase. In fact, the spindle assembly checkpoint may block the metaphase--~anaphase transition specifically by targeting this ubiquitination/degradation system (Rudner and Murray, pp 773-780), and, in doing so, may stabilize

proteins that need to be destroyed in order to achieve chromatid separation. The targeting of B-type cyclins by the same system has already been shown to be required for exit from mitosis. Thus, progression through the cell cycle appears to require both the synthesis and the proteolysis of key regulatory proteins. Determining how the coordination between these two opposing but interregulated processes is accomplished is one of the major current challenges in the cell cycle field.

References 1.

Pardee AB: G1 events and regulation of cell proliferation. Science 1989, 246:603-608.

2.

Deshaies RJ: The self-destructive personality of a cell cycle in transition. Curt Opin Cell Bio/1995, 7:781-789.