676

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle Susanna V Ekholm* and Steven I Reed
Cyclin-dependent kinases are the key regulators of cell-cycle transitions. In mammalian cells, Cdk2, Cdk4, Cdk6 and associated cyclins control the G1 to S phase transition. Because proper regulation of this transition is critical for an organisms survival, these protein kinases are exquisitely regulated at different mechanistic levels and in response to a large variety of intrinsic and extrinsic signals.
Addresses *Department of Oncology/Pathology, Cancer Center Karolinska, Karolinska Insitutet, 17176 Stockholm, Sweden Department of Molecular Biology, MB-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA; e-mail: sreed@scripps.edu Current Opinion in Cell Biology 2000, 12:676684 0955-0674/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. Abbreviations Cdk cyclin-dependent kinase CKIs Cdk inhibitors HDAC histone deacetylase PI 3-kinase phosphatidylinositol 3-kinase pRB retinoblastoma susceptibility protein

requisite positive regulatory subunit (cyclin), the first level of regulation therefore is cyclin availability. Another layer of control derives from inhibitory phosphoryation of the Cdk and binding of inhibitor proteins (CKIs; Cdk inhibitors), both of which have broad regulatory significance. As will be discussed below, the dominant mode of regulation for a particular Cdk is highly context dependent.
Cyclin-dependent kinase structure

The determination of the structures, through X-ray diffraction crystallography, of Cdks, cyclins, Cdkcyclin complexes, and CdkCKI complexes has revealed much about their regulation. Cdks conform to the basic protein kinase structural motif, which consists of a -sheet-rich amino-terminal lobe and and a largely -helical carboxy-terminal lobe, with the active site falling between the two ([10,11]). Both the positive regulatory impact of cyclin binding, as well as the mechanism whereby the two classes of mammalian CKIs exert inhibitory action, are now well understood [10,11].
Cyclin-dependent kinase function

Introduction
It has been approximately 15 years since the significance of the cyclin-dependent kinase (Cdk) motif was established for cell-cycle control. During the ensuing years much has been learned concerning the structure and function of this evolutionarily conserved class of enzymes. New insights continue to arise as knowledge of cell-cycle control becomes more comprehensive and as research tools become more sophisticated. This review provides a general summary of of G1 Cdk regulation in mammalian cells, focusing particularly on the most recent advances.
Cyclin-dependent kinases

Surprisingly little is known about the biological targets of Cdk activity. Even in yeast, using sophisticated genetic analysis, only a few key targets have been identified. This is probably due to the output of Cdk function being achieved by the phosphorylation of many proteins and not by the activation of linear signal cascades that are easy to identify both genetically and biochemically. One notable exception is the mammalian retinoblastoma susceptibility protein, pRb. pRb is a component of a transcriptional repression module that targets many genes whose products function during S phase or at the G1/S transition. Phosphorylation of pRb by G1 Cdks near the G1/S boundary relieves this repressive activity and allows transcription of S phase genes (see below) [12,13].
G1 cyclin-dependent kinases

Cdks were discovered initially by genetic analysis of the cell cycle in yeast and through the analysis of M (mitosis) phase inductive activities in frog and marine invertebrate eggs and early embryos. However, within a relatively short time, Cdks were found to be a universal hallmark of the eukaryotic cell cycle, and it is now clear that Cdks, either directly or indirectly, control the major cell-cycle transitions and phases in all eukaryotic organisms (for basic reviews on Cdks see [19]). Because of their central regulatory roles in cell proliferation, Cdks themselves are subject to many modes and levels of regulation in response to both intracellular and extracellular signals [6,7]. As Cdks form a binary system composed of an inactive catalytic subunit (Cdk) and a

In mammalian cells, there are two classes of Cdks that function at the G1/S phase transition [5,9,14]. Cdk4 and its close relative Cdk6 are driven by three D-type cyclins: D1, D2 and D3. The primary target of these activities is pRb and related proteins [15]. Cyclin E accumulates very close to the G1/S phase transition and specifically activates Cdk2. Although cyclin-ECdk2 has a secondary role in phosphorylating pRb [12,13], after cyclin-DCdk4/6 the critical target(s) of cyclin-ECdk2 in regulation of S phase is not known.

Regulation of cyclin-dependent kinase activity by cyclins

Cyclin abundance oscillates during the cell cycle as a result of programmed synthesis and degradation, thereby assuring

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle Ekholm and Reed

677

Figure 1 (a) Transcription (b) Translation (c) Proteolysis (f) Phosphorylation P P Cyclin D Cdk4/6 (e) INK4 (g) Proteolysis Cdk4/6 (h) Inhibitory phosphorylation Cip/Kip
Current Opinion in Cell Biology

Cyclin D

(d) Cdc37HSP90 Cip/Kip

P Target

Regulation of cyclin-DCdk4/6. D-type cyclins have been shown to be regulated at the level of biosynthesis, both (a) transcriptionally [22,23,24,25,26,2729] and (b) translationally [32,33,34,35]. The steady state level of free cyclin D is additionally controlled by (c) ubiquitin-dependent proteolysis [40]. Once synthesized, the functional association of D-type cyclins with Cdk4 or Cdk6 depends on (d) the Cdc37/Hsp90 chaparonin [48,49] as well as (d) Cip/Kip

inhibitors [50,98,99]. Functional association between D-type cyclins and Cdk4 or Cdk6 is also regulated negatively (e) by INK4 inhibitors [10,11]. Once assembled with D-type cyclins, Cdk4 and Cdk6 require (f) T-loop phosphorylation for activation [10,11]. Activity is also controlled at the level of (g) D-type cyclin proteolysis [39]. Cyclin D/Cdk4 complexes have been shown to be subjected to (h) inhibitory phosphorylation [103] as well as inhibition by Cip/Kip inhibitors [54].

a limited window of Cdk activation. Overexpression of D-type cyclins or cyclin E during early G1 leads to premature S phase entry [1618], suggesting that the G1 cyclins are at least partially rate limiting for S phase entry and confirming that regulation of Cdk activity by cyclin accumulation has biological consequences.
Regulation of cyclin synthesis

Once synthesized, cyclin D promotes cell-cycle progression by two complementary mechanisms, one direct (Cdk4/6 activation) and one indirect. Stimulation of progression into S phase by c-Myc depends largely on activation of cyclin-ECdk2 via transcription of cyclin D1 and/or D2 and a mechanism known as CKI exchange [22,23] (see below). Cyclin E expression is more periodic than that of D-type cyclins. Cyclin E mRNA and protein begin to accumulate in late G1, peak at the G1/S transition and are downregulated during S phase [9,30]. Cyclin E transcription is activated when pRb is hyperphosphorylated and no longer exerts repression via E2F/DP, the transcription factor complex that targets pRb-mediated repression. Consistent with this, the cyclin E promoter contains several putative binding sites for E2F. One of them, a variant E2F-binding site, was recently identified as a cyclin E repressor module. This site mediates transcriptional repression by binding of a large E2F4-containing repressor complex ([31]; see review by William Harbour and Douglas Dean on pp 685690 of this issue). Interestingly, expression of cyclin E and A is regulated differentially by the same large repressor complex containing pRb, hSWI/SNF and HDAC (histone deacetylase) [12]. Cyclin-DCdk4-dependent phosphorylation disrupts the association with HDAC, leading to cyclin E induction and progression into S phase. The resulting complex, now containing only pRb and hSWI/SNF, still represses transcription of cyclin A and Cdk1, thus controlling the temporality of cyclin and Cdk expression [12]. D-type cyclin availability is also regulated at the translational level through the phosphatidylinositol 3-kinase (PI 3-kinase) pathway [32,33]. Mitogens increase the rate of cyclin D1 translation by activation of the translation initiation

G1 progression depends on the sustained expression of D-type cyclins, which, in turn, depends on continuous mitogenic stimulation, suggesting that D-type cyclins provide a link between mitogen signaling and the cell-cycle machinery. The three highly homologous D-type cyclin isoforms are differentially expressed in an organ-specific and tissue-specific manner [19]. Consistent with this, functional analysis of the D-type cyclin genes has revealed marked differences in the elements regulating their transcription. However, analysis of cyclin D nullizygous mice suggests a surprising degree of functional redundancy [20,21]. In the context of mitogen stimulation, the D-type cyclin genes have been shown to be transcriptionally induced by c-Myc [22,23], AP-1 [24,25,26] and NF-B [27,28]. Transcriptional output is a function of both inductive and repressive activities [24,29]. For example, the transcription factor AP-1 consists of Jun and Fos proteins [24,25,26], and c-Jun and JunB have been demonstrated to exert opposing effects on cyclin D1 transcription: c-Jun was shown to induce it and JunB to repress it. Thus, variation in the ratio of c-Jun to JunB and regulatory phosphorylation of these factors are critical for modulating cyclin D1 expression during G1 [26]. Interestingly, similar but opposite AP1-isoform-dependent transcriptional modulation has been suggested for the Cdk4/6 inhibitor p16 (see below).

678

Cell multiplication

Figure 2

(a) Transcription

(b) Proteolysis (e) Phosphorylation P P Cyclin E Cdk2 (f) Phosphorylation Cip/Kip

Current Opinion in Cell Biology

Cyclin E

(d) CCT

P Target

Cdk2 (c) Proteolysis

Regulation of cyclin-ECdk2. Cyclin E accumulation has been shown to be regulated at the level of (a) transcription [9,12,30,31] and degradation. Degradation pathways differentially target (b) free cyclin E [45] and (c) Cdk2-bound cyclin E [41,42,43,44]. Maturation and binding of

cyclin E to Cdk2 also requires the activity of the (d) CCT chaparonin complex [46]. Cyclin E-bound Cdk2 must be activated by (e) T-loop phosphorylation [10,11]. Cyclin E/Cdk2 complexes can be regulated by (f) inhibitory phosphorylation [98,99] and Cip/Kip inhibitors [50,52].

factor eIF-4E [34], whereas antiproliferative agents decrease cyclin D1 synthesis through the inactivation of eIF-4E [35]. In support of these findings, the stress-induced unfolded protein response pathway was found to block translation of cyclin D1 mRNA, despite continuous mitogenic stimulation through phosphorylation and inactivation of eIF-4E [36].
Regulation of cyclin degradation

Cyclins are unstable proteins that are degraded via the ubiquitin/proteasome pathway (reviewed in [37]). Phosphorylation-dependent ubiquitination and the subsequent degradation of proteins by the 26S proteasome relies on the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a proteinubiquitin ligase (E3). The SCF (Skp1Cullin-1F-box) family of protein-ubiquitin ligases has been implicated in the phosphorylation-dependent ubiquitination of G1 cyclins, particularly in yeast [37]. SCF complexes have a substrate specificity factor known as an F-box protein [38]; however, the involvement of SCF in ubiquitination of mammalian G1 cyclins is not clear. Recent evidence suggests that distinct pathways for ubiquitination of G1 cyclins operate depending on whether the cyclin is bound to Cdk or not. The direct involvement of an SCF complex in cyclin D1 degradation has not been demonstrated, although degradation is dependent on phosphorylation of threonine 286 by glycogen synthase kinase 3 (GSK3), an event that is stimulated when cyclin D1 is bound to Cdk4 [39]. Recently, a novel pathway for ubiquitination and degradation of free cyclin D1, which is independent of phosphorylation, has been identified [40]. Cyclin E degradation is also mediated via distinct pathways. Degradation of Cdk2-bound cyclin E is dependent on phosphorylation on threonine 380 [41,42], consistent with involvement of the SCF pathway, but evidence for a direct interaction between an SCF complex and phoshorylated cyclin E has yet to be presented. Also

consistent with an SCF-dependent mechanism, Cullin-1/ mice arrested in early development and embryonic cells were found to contain high levels of cyclin E [43,44]. A distinct mechanism involving a non-SCF relative of Cul1, Cul3, has been demonstrated for degradation of free cyclin E and is not dependent on phosphorylation [45]. However, cyclin E accumulates in only a few cell types in embryos of Cul3/ mice [45], leaving the role of the Cul3 pathway in cyclin E degradation unclear.
Folding

Cyclin E accumulation also depends on the activity of the cytosolic chaperonin CCT, which mediates the ATPdependent folding of newly translated cyclin E into a mature form that can associate with Cdk2 [46]. Whether this essential process has regulatory significance or merely corresponds to a housekeeping function is not known.
Association with cyclin-dependent kinases

The first step in the activation of cyclinCdk complexes is the association of the cyclin subunit with a Cdk partner. Assembly of ectopically expressed Cdk4 and cyclin D3 in human fibroblasts requires a mitogen-dependent step, which is likely to involve an assembly factor [47]. Although the identity of this essential factor is not known, the chaperonin complex Cdc37Hsp90 specifically interacts with Cdk4 and Cdk6 and facilitates assembly of cyclin-D1Cdk4 complexes in vitro [48,49]. In the absence of functional Hsp90 in vivo, Cdk4 is rapidly turned over, suggesting a role for Cdc37Hsp90 in regulating Cdk activity and cell-cycle progression [49]. Paradoxically, the Cip/Kip family of CKIs also has a role in cyclin-DCdk4/6 assembly [50] (see below).

Cyclin-dependent kinase inhibitors

Recent research has identified two classes of CKIs in vertebrates. Other proteins have Cdk inhibitory properties,

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle Ekholm and Reed

679

but bona fide in vivo roles as Cdk inhibitors remain to be established [51]. The two classes are the Cip/Kip family and the INK4 family. The Cip/Kip family is composed of three members: p21WAF1/CIP1, p27KIP1, and p57KIP2. The INK4 family is composed of four members: p15, p16, p18 and p19 [50,52]. This classification is based on their structure as well as their Cdk affinity. The Cip/Kip CKIs have amino-terminal Cdk-inhibitory domains and structurally divergent carboxy-terminal domains for which less functional information is available. The three-dimensional structure of a ternary complex composed of the Cdkinhibitory domain of p27, Cdk2, and the amino-terminal (Cdk-activating) half of cyclin A was determined recently [53]. It revealed that inhibition is achieved by first anchoring the inhibitory polypeptide to the cyclin, extending across the amino-lobe of the Cdk, and finally invading the active site, culminating in displacement of critical -strands and occupation of the adenine-binding pocket of the substrate-binding site [53]. Although the structures of p21 and p57 have not yet been determined, high primary structure homology in key regions of the respective polypeptides strongly suggests analogous inhibitory mechanisms. Cip/Kip inhibitors are considered broad spectrum CKIs in that they can bind to and inhibit both cyclin-DCdk4/6 kinases, as well as cyclin-E/ACdk2, although it has been reported that the efficiency of Cdk4/6 inhibition may vary for the different Cip/Kip inhibitors [54]. In addition Cip/Kip inhibitors may have a paradoxical role in activating these kinases (see below). The INK4 inhibitors share a common structural motif and mechanism of inhibition. They consist of either four (p15 and p16) or five (p18 and p19) repeating structural units known as ankyrin repeats. Ankyrin repeats form a concave structure composed of stacked L-shaped domains (each ankyrin repeat), which in the case of INK4 inhibitors bind across the back (non-catalytic) side of the target kinase [55,56]. The result of this is that the Cdk amino-lobe is rotated by 15 relative to the carboxy-lobe around a flexible axis, forcing Cdk into a conformation that cannot support catalysis [55,56]. In addition, INK4-bound Cdks cannot bind to cyclin and thus are isolated as CdkINK4 heterodimers [55,56]. Finally, INK4 inhibitors are narrow-spectrum CKIs; they only bind to and inhibit Cdk4 and Cdk6. The differential specificities of the different classes of CKIs have important implications for cell-cycle regulation (see below).
Regulation of CKI synthesis

and stimulus-specific manner, frustrating any attempt at generalization. Nevertheless, a few well elucidated examples serve to illustrate how CKI gene expression is controlled to meet regulatory demands. The archetypal mammalian CKI is p21. It was first discovered on the basis of its p53-responsive expression [57] and then through binding and inhibiting Cdk2 [58]. p21 is largely responsible for p53-dependent G1 arrest in response to genotoxic stress [59]. Induction and activation of p53 leads to transcriptional induction of p21 owing to strong p53 response elements in the p21 gene promoter. The subsequent accumulation of p21 then leads to binding to and inactivation of G1 Cdks. This model is confirmed in cells derived from p21 nullizygous mice, where most of the G1 arrest response to DNA damage is lost [60,61]. However, the role of p53 may be more complicated: it has been reported that p53 also regulates p21 at the level of mRNA stability [62]. It is clear that p21 has many roles in cell-cycle control beyond its function as an effector of p53 responses to genotoxic stress. p53-independent expression of p21 occurs in various tissues throughout embryogenesis and is associated with cell-cycle exit in the context of terminal differentiation [63,64], for example in the process of myotube formation. p21 is induced as myocytes become post-mitotic and fuse to form syncytial myotubes during muscle development [65,66], and analysis of Cdk inactivation during myotube formation is at least consistent with a role for p21. However, p21 nullizygous mice are not ostensibly defective in muscle development, suggesting that p21 by itself cannot be central to myocyte terminal differentiation [60,61]. This finding underscores the fact that cell-cycle control is often mechanistically redundant. Consistent with this idea, p21 p57 doubly nullizygous embryos cannot form myotubes [67]. Like p21, p57 shows a distinct tissue-specific pattern of expression during development, similarly suggesting a role in cell-cycle exit during terminal differentiation [68]. No simple rule can be established for transcriptional control of INK4 inhibitors. Yet clearcut examples of transcriptional regulation in specific cell types have been reported. The best characterized transcriptional response of an INK4 inhibitor is the induction of p15 (INK4b) in response to the cytokine TGF in epithelial cells. TGF treatment of epithelial cells mobilizes transcription factors of the Smad family by a well elucidated receptor-dependent pathway [69]. Surprisingly, the p15 promoter is Smad responsive but does not contain cononical elements for Smad binding, and therefore the precise mechanism of transcriptional activation remains to be determined [70,71]. However, cells deficient in the p15 response to TGF are still capable of cytokine-mediated arrest by downregulation of the Cdk phosphatase CDC25A [72], (see below), again suggesting a high degree of redundancy in cell-cycle control.

CKIs were initially proposed to accumulate in response to a cells need to cease dividing. This has been confirmed in many situations, ranging from checkpoint control in response to DNA damage, to entry into a postmitotic terminally differentiated state. However, it is difficult to draw strong conclusions concerning the molecular basis for regulation of CKI levels. The reason is that although all cells use the same repertoire of CKIs, they apparently regulate them differently, in a cell-type

680

Cell multiplication

p16 (INK4a) accumulates in many human cells during clonal senescence. Whether this is important as a mechanism of cell-cycle control in senescence is not clear (see review by Robert Weinberg and colleagues, pp 705709 of this issue). In mice it has been suggested that a protein derived from the same coding region but via an alternative reading frame, ARF, is critical for senescence and not p16 [73]. However, senescence appears to be mechanistically different in humans and mice. The human p16 promoter is highly methylated and inactivated in young cells [74,75,76]. However, progressive demethylation of the p16 promoter as cells age is associated with increasing p16 expression [74,75,76]. Thus, demethylation of the genome with age may constitute a mechanism for coupling aging to the induction of mediators of senescence. Unique among the INK4 inhibitors, p16 is a tumor suppressor associated particularly with familial and sporadic melanoma [77]. It is not yet clear why p16 should be particularly important in preventing malignancy, but the p16 promoter has AP-1 response elements that are highly responsive to JunB-containing complexes [78]. Furthermore, UV irradiation leads to activation of AP-1 complexes containing JunB and concomitant induction of p16, suggesting that p16 induction is a critical part of the checkpoint response to UV damage [79,80]. Interestingly, the same AP-1 complexes that induce p16 repress cyclin D1, potentially reinforcing a cell-cycle block. Melanocytes may be particularly susceptible to impairment of this checkpoint. Transcriptional control is not the universally preferred mode for regulation of CKI biosynthesis. In fact, p27 appears to be regulated primarily by translational control [81,82]. Under most conditions, transcription of the p27 gene is constitutive, and analysis of the p27 promoter indicates that it is driven by the ubiquitous transcription factor Sp1 [8385]. Although p27 levels and rate of synthesis increase dramatically as fibroblasts exit the cell cycle as a result of serum withdrawal or contact inhibition, p27 mRNA levels remain constant [81]. However, induction of p27 mRNA has also been reported in some model differentiation systems [86,87].
Regulation of CKI proteolysis

S phase and G2 coupled to a constitutively high rate of ubiquitin-mediated proteolysis [92]. Thus, low levels during G1 assure that active cyclin-DCdk4/6 complexes can assemble during G1 when they are required.
Regulation of cyclin-dependent kinases by CKI exchange

Cyclin-DCdk4/6 complexes have a dual role. First, in promoting cell-cycle progression by phosphorylating pRb and related negative cell-cycle regulators, and second, as a reservoir for Cip/Kip inhibitors. For this latter role, accumulation of D-type cyclins promotes the G1/S phase transition specifically by sequestering Cip/Kip inhibitors away from cyclin-ECdk2 and thereby activating it. Conversely, the disruption of cyclin-DCdk complexes, for example, via the accumulation of INK4 inhibitors, drives Cip/Kip inhibitors to cyclin-ECdk2, leading to inhibition. This secondary effect appears to be essential for tight cell-cycle regulation. D-type cyclin mediated exchange is the primary mechanism for activation of cyclin-ECdk2 by c-Myc. Expression of c-Myc leads to removal of p27 from, and concomitant activation of, cyclin-ECdk2 complexes. The demonstration that the cyclin D1 and D2 genes are transactivated by c-Myc, that the resulting cyclin D accumulation leads to redistribution of p27 to cyclin-DCdk complexes and that cyclin D2 nullizygous cells do not activate cyclin-ECdk2 complexes efficiently in response to c-Myc expression confirms this model [22,23,93]. Also consistent with a fundamental role for CKI exchange, cells from Cdk4 nullizygous mice are slow to enter the cell cycle because of an impaired ability to activate cyclin-ECdk2 complexes by p27 redistribution to cyclin-DCdk4 complexes [94]. The forced redistribution of Cip/Kip inhibitors to cyclin-ECdk2 complexes via the action of INK4 inhibitors is best exemplified by the response of epithelial cells to the cytokine TGF, although this phenomenon can be invoked under any circumstance where an INK4 inhibitor is induced. TGF stimulates the transcriptional induction of p15, which then forms stable complexes with Cdk4 and Cdk6, preventing them from interacting with Cip/Kip inhibitors and driving the latter to associate with and inhibit cyclin-ECdk2 [95]. Reconstruction experiments using a dominant-negative allele of Cdk4 or a cell line nullizygous for p21 confirm that inhibition of cyclin-DCdk4/6 complexes is not sufficient to account for the tight cell-cycle arrest conferred by INK4 inhibitor expression and that Cip/Kip exchange is an essential mechanistic component [96,97].
Activation of cyclin-dependent kinases by Cip/Kip inhibitors

For p27, proteolysis is one of the key modes of regulation. p27 is targeted for degradation via the ubiquitin/proteasome pathway. In particular, the protein-ubiquitin ligase known generically as SCF (discussed above) specifically ubiquitinates p27 that has been phosphorylated by cyclin-ECdk2 [88,89,90]. In addition, the specificity factor, or F-box protein, that recognizes p27, Skp2, is synthesized periodically at the G1/S phase boundary [91]. Thus, both the accumulation of cyclin E and concomitant activation of Cdk2, as well as the accumulation of the SCF component Skp2, serve to couple p27 proteolysis to cells committed to entering S phase (see also Update). Of the INK4 inhibitors, only p19 is periodic throughout the cell cycle and this is achieved by transcription during

Paradoxically, Cip/Kip inhibitors have been associated with Cdk activation as well as inhibition. Cip/Kip inhibition of cyclin-DCdk4 complexes is relatively inefficient [54]. At the same time, assembly of cyclin-DCdk4 complexes in vitro leads largely to inactive dimers [98]. However, presumably because of their ability to bridge between cyclin and Cdk, Cip/Kip inhibitors can serve as chaperonins, facilitating

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle Ekholm and Reed

681

attainment of an active conformation [98]. These in vitro observations have recently been confirmed in vivo using fibroblasts from mice nullizygous for both p21 and p27. In the absence of both CKIs, virtually no cyclin-DCdk4 complexes were detectable [99]. Yet, surprisingly, the effects on the cell cycle were minimal, suggesting that the absence of inhibitors compensated for the inefficient assembly of cyclin-DCdk4 complexes [99].

require ubiquitination. Mutant versions of p21 that cannot be ubiquitinated in vivo remain unstable and increase in abundance upon proteasome inhibition [105].

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest

1. 2. 3. 4. 5. 6. 7. 8. 9. Hunt T: Cyclins and their partners: from a simple idea to complicated reality. Semin Cell Biol 1991, 2:213-222. Reed SI: The role of p34 kinases in the G1 to S-phase transition. Annu Rev Cell Biol 1992, 8:529-561. Norbury C, Nurse P: Animal cell cycles and their control. Annu Rev Biochem 1992, 61:441-470. Solomon MJ: Activation of the various cyclin/cdc2 protein kinases. Curr Opin Cell Biol 1993, 5:180-186. Sherr CJ: G1 phase progression: cycling on cue. Cell 1994, 79:551-555. Morgan DO: Principles of CDK regulation. Nature 1995, 374:131-134. Nigg EA: Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays 1995, 17:471-480. Pines J: Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J 1995, 308:697-711. Reed SI: Control of the G1/S transition. Cancer Surv 1997, 29:7-23.

Regulation of cyclin-dependent kinase phosphorylation

Inhibitory phosphorylation has been best characterized for Cdk1 in the context of the G2/M-phase transition (see review by Catherine Takizawa and David Morgan on pp 658665 of this issue). However, Cdk2 and Cdk4 are also phosphorylated on tyrosine 15 and 17, respectively, and phosphatase treatment in vitro leads to hyperactivation of Cdk2 kinase activity. Mammalian cells contain three specialized phosphatases that reverse inhibitory Cdk phophorylation: these are CDC25A, B and C. On the basis of expression patterns of the three, CDC25A is assumed to be responsible for Cdk2 activation [100]. Furthermore, ectopic expression of CDC25A accelerates the G1/S phase transition by dephosphorylating cyclin-ECdk2 complexes ahead of schedule [101]. Finally, CDC25A is activated late in G1, corresponding to the time when cyclin-ECdk2 complexes become active [101]. Thus, in addition to binding of Cip/Kip inhibitors before the G1/S phase transition, phosphorylation of Cdk2 is used to accumulate and maintain cyclin-ECdk2 complexes in a pre-active state. Regulation of CDC25A is critical for the G1 response to DNA damage. Mammalian cells respond to UV or ionizing radiation by rapid ubiquitin/proteasome-mediated degradation of CDC25A [102]. This response precedes the p53-dependent accumulation of p21 and is essential for maximal DNA repair and survival. Both Cdk2 and Cdk4 may be targets of CDC25A regulation. In quiescent cells, tyrosine 17 of Cdk4 is phosphorylated and UV irradiation prevents dephosphorylation during cell cycle re-entry [103], possibly by downregulation of CDC25A. TGF and interferon signaling also lead to CDC25A downregulation, although the mechanisms remain to be determined [72,104].

10. Pavletich NP: Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol 1999, 287:821-828. 11. Endicott JA, Noble ME, Tucker JA: Cyclin-dependent kinases: inhibition and substrate recognition. Curr Opin Struct Biol 1999, 9:738-744. 12. Zhang HS, Gavin M, Dahiya A, Postigo AA, Ma D, Luo RX, Harbour JW, Dean DC Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell 2000, 101:79-89. pRb, in association with histone deacetylase (HDAC) and hSWI/SNF is shown to form a transcriptional repression complex that inhibits transcription of cyclin E and cyclin A during G1 phase. The cyclin E gene is induced when HDAC is released from the complex, which is a consequence of Rb phosphorylation by cyclin-Dcdk4. However, transcription of cyclin A and Cdk1 is repressed by this complex until pRb is phosphorylated by cyclin-ECdk2 in S phase. These complexes seem to regulate the order of cyclin and Cdk expression. 13. Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC: Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 1999, 98:859-869. This paper shows that progressive phosphorylation of pRb by various Cdks during G1 causes sequential allosteric changes that sequentially block various pRb-mediated inhibitory functions. 14. Draetta GF: Mammalian G1 cyclins. Curr Opin Cell Biol 1994, 6:842-846. 15. Grana X, Garriga J, Mayol X: Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth. Oncogene 1998, 17:3365-3383. 16. Ohtsubo M, Roberts JM: Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science 1993, 259:1908-1912. 17. Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF, Sherr CJ: Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 1993, 7:1559-1571.

Conclusions
Research over the past several years has yielded an increasingly detailed perspective of Cdk structure and function. Yet understanding their role in the large number of distinct cell types, all with differing cell-cycle regulatory requirements and signaling modes, remains a daunting challenge. For this reason, in coming years, cell-cycle research is likely to become increasingly dependent on whole-animal genetics, particularly nullizygous and knock-in mice and reagents derived from them.

18. Resnitzky D, Gossen M, Bujard H, Reed SI: Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 1994, 14:1669-1679. 19. Sherr CJ: Mammalian G1 cyclins. Cell 1993, 73:1059-1065. 20. Fantl V, Stamp G, Andrews A, Rosewell I, Dickson C: Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev 1995, 9:2364-2372.

Update
Unlike ubiquitin-dependent targeting of p27 for turnover, proteasomal degradation of p21 does not apparently

682

Cell multiplication

21. Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 1995, 82:621-630. 22. Perez-Roger I, Kim SH, Griffiths B, Sewing A, Land H: Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1). EMBO J 1999, 18:5310-5320. In this study and [23], c-Myc-induced sequestration of p21 and p27 and activation of cyclinEcdk2 complexes are shown to be mediated via induction of cyclin D1 and/or cyclin D2. Primary cells from cyclin D1 / and D2/ mouse embryos were insensitive to c-Myc, suggesting that the CKI sequestration function of D-type cyclins is essential for Myc-induced cell-cycle progression. 23. Bouchard C, Thieke K, Maier A, Saffrich R, Hanley-Hyde J, Ansorge W, Reed S, Sicinski P, Bartek J, Eilers M: Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27. EMBO J 1999, 18:5321-5333. See annotation [22]. 24. Herber B, Truss M, Beato M, Muller R: Inducible regulatory elements in the human cyclin D1 promoter. Oncogene 1994, 9:2105-2107. 25. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell RG: Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 1995, 270:23589-23597. 26. Bakiri L, Lallemand D, Bossy-Wetzel E, Yaniv M: Cell-cycle dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. EMBO J 2000, 19:2056-2068. This paper shows that cyclin D1 expression is modulated during G1 by variations in c-Jun and JunB ratios and phosphorylation. c-Jun activates cyclin D1 whereas JunB represses transcription. These findings clarify the role of AP-1 in G1 progression. 27. Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS Jr: NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999, 19:5785-5799.

37.

Tyers M, Jorgensen P: Proteolysis and the cell cycle: with this RING I do thee destroy. Curr Opin Genet Dev 2000, 10:54-64.

38. Krek W: Proteolysis and the G1-S transition: the SCF connection. Curr Opin Genet Dev 1998, 8:36-42. 39. Diehl JA, Cheng M, Roussel MF, Sherr CJ: Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998, 12:3499-3511. This paper identifies GSK-3 as the kinase responsible for phosphorylating cyclin D1 in complex with Cdk4 and targeting it for proteolysis. This is in contrast to cyclin E in complex with Cdk2, which is phosphorylated by Cdk2 itself, presumably by autophosphorylation. 40. Germain D, Russell A, Thompson A, Hendley J: Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286. J Biol Chem 2000, 275:12074-12079. A pathway for ubiquitination and degradation of free cyclin D1, which is independent of phosphorylation, is identified. These data suggest that Cdk-bound cyclin D1 and free cyclin D1 are targeted for ubiquitination and degradation by distinct pathways. 41. Clurman BE, Sheaff RJ, Thress K, Groudine M, Roberts JM: Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev 1996, 10:1979-1990. 42. Won KA, Reed SI: Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E. EMBO J 1996, 15:4182-4193. 43. Wang Y, Penfold S, Tang X, Hattori N, Riley P, Harper JW, Cross JC, Tyers M: Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E. Curr Biol 1999, 9:1191-1194. This study and [44] show that Cul1/ mice arrest in early embryogenesis with elevated levels of cyclin E protein although the level of mRNA in all cell types is not increased, consistent with a role for SCF in cyclin E proteolysis. 44. Dealy MJ, Nguyen KV, Lo J, Gstaiger M, Krek W, Elson D, Arbeit J, Kipreos ET, Johnson RS: Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E. Nat Genet 1999, 23:245-248. See annotation [43]. 45. Singer JD, Gurian-West M, Clurman B, Roberts JM: Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes Dev 1999, 13:2375-2387. In this study, Cul-3 was identified as a protein that could associate with and target only free cyclin E for ubiquitination and degradation by a phosphorylation-independent pathway. Cul-3/ embryos arrest in early development but elevated levels of cyclin E were detected in only a few cell types. 46. Won KA, Schumacher RJ, Farr GW, Horwich AL, Reed SI: Maturation of human cyclin E requires the function of eukaryotic chaperonin CCT. Mol Cell Biol 1998, 18:7584-7589. The eukaryotic chaperonin CCT was shown to associate with newly synthesized cyclin E. CCT is essential for the folding or maturation of cyclin E in vivo, a process required for binding to cdk2. 47. Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato JY: D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol 1994, 14:2066-2076.

28. Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M: NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol 1999, 19:2690-2698. 29. Watanabe G, Albanese C, Lee RJ, Reutens A, Vairo G, Henglein B, Pestell RG: Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1. Mol Cell Biol 1998, 18:3212-3222. 30. Keyomarsi K, Herliczek TW: The role of cyclin E in cell proliferation, development and cancer. Prog Cell Cycle Res 1997, 3:171-191. 31. Le Cam L, Polanowska J, Fabbrizio E, Olivier M, Philips A, Ng Eaton E, Classon M, Geng Y, Sardet C: Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex. EMBO J 1999, 18:1878-1890. The authors identify a new E2F4-containing transcription repressor element that controls repression of cyclin E during G0/G1. This repressor activity is still present in Rb/cells. 32. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N: Cyclin D expression is controlled posttranscriptionally via a phosphatidylinositol 3-kinase/Aktdependent pathway. J Biol Chem 1998, 273:29864-29872. 33. Takuwa N, Fukui Y, Takuwa Y: Cyclin D1 expression mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol Cell Biol 1999, 19:1346-1358. This study and [32] show that cyclin D1 expression is controlled posttranscriptionally by the PI-3 kinase/Akt pathway via regulation of translation. 34. Sonenberg N, Gingras AC: The mRNA 5 cap-binding protein eIF4E and control of cell growth. Curr Opin Cell Biol 1998, 10:268-275. 35. Aktas H, Fluckiger R, Acosta JA, Savage JM, Palakurthi SS, Halperin JA: Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole. Proc Natl Acad Sci USA 1998, 95:8280-8285. 36. Brewer JW, Hendershot LM, Sherr CJ, Diehl JA: Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proc Natl Acad Sci USA 1999, 96:8505-8510.

48. Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet C, Draetta GF, Gyuris J: Interaction between Cdc37 and Cdk4 in human cells. Oncogene 1997, 14:1999-2004. 49. Stepanova L, Leng X, Parker SB, Harper JW: Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4. Genes Dev 1996, 10:1491-1502. 50. Sherr CJ, Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999, 13:1501-1512. 51. Coats S, Whyte P, Fero ML, Lacy S, Chung G, Randel E, Firpo E, Roberts JM: A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells. Curr Biol 1999, 9:163-173. 52. Vidal A, Koff A: Cell-cycle inhibitors: three families united by a common cause. Gene 2000 247:1-15. 53. Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP: Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 1996, 382:325-331. 54. Blain SW, Montalvo E, Massague, J: Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) with cyclin A-cdk2 and cyclin D2-cdk4. J Biol Chem 1997, 272:25863-25872.

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle Ekholm and Reed

683

55. Russo AA, Tong L, Lee JO, Jeffrey PD, Pavletich NP: Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a. Nature 1998, 395:237-243. This study and [56] show the structure of Cdk6 bound to INK4 inhibitors and elucidates the mode of inhibition. 56. Brotherton DH, Dhanaraj V, Wick S, Brizuela L, Domaille PJ, Volyanik E, Xu X, Parisini E, Smith BO, Archer SJ et al.: Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d. Nature 1998, 395:244-250. See annotation to [55]. 57. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B: WAF1, a potential mediator of p53 tumor suppression. Cell 1993, 75:817-825.

74. Brenner AJ, Stampfer MR, Aldaz CM: Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene 1998, 17:199-205. 75. Foster SA, Wong DJ, Barrett MT, Galloway DA: Inactivation of p16 in human mammary epithelial cells by CpG island methylation. Mol Cell Biol 1998, 18:1793-1801. 76. Wong DJ, Foster SA, Galloway DA, Reid BJ: Progressive region specific de novo methylation of the p16 CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest. Mol Cell Biol 1999, 19:5642-5651. This study, as well as [74] and [75], shows that demethylation is associated with p16 expression and senescence in aging human mammary epithelial cells and that progressive methylation is associated with escape from senescence. 77. Castellano M, Parmiani G: Genes involved in melanoma: an overview of INK4a and other loci. Melanoma Res 1999, 9:421-432.

58. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclindependent kinases. Cell 1993, 75:805-816. 59. Dulic V, Kaufmann, WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI: p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 1994, 76:1013-1023. 60. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P: Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 1995, 82:675-684. 61. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ: Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 1995, 377:552-557. 62. Gorospe M, Wang X, Holbrook NJ: p53-dependent elevation of p21waf1 expression by UV light is mediated through mRna stabilization and involves a vanadate-sensitive regulatory system. Mol Cell Biol 1998, 18:1400-1407. 63. Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, Jacks T: p53-dependent and independent expression of p21 during cell growth, differentiation, and Dna damage. Genes Dev 1995, 9:935-944. 64. Parker SB, Eichele G, Zhang P, Rawls A, Sands AT, Bradley A, Olson EN, Harper JW, Elledge SJ: p53-independent expression of p21cip1 in muscle and other terminally differentiating cells. Science 1995, 267:1024-1027. 65. Halevy O, Novitch BG, Spicer DB, Skapek SX, Rhee J, Hannon GJ, Beach D, Lassar AB: Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 1995, 267:1018-1021. 66. Guo K, Wang J, Andres V, Smith RC, Walsh K: MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation. Mol Cell Biol 1995, 15:3823-3829. Zhang P, Wong C, Liu D, Finegold M, Harper JW, Elledge SJ: p21(Cip1) and p57(Kip2) control muscle differentiation at the myogenin step. Genes Dev 1999, 13:213-224. This study shows that the Cip/Kip inhibitors p21 and p57 have redundant functions in myogenesis. It explains the lack of a muscle phenotype in p21 nullizygous mice. 68. Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, Thompson JC, Silverman A, Harper JW, DePinho RA, Elledge SJ: Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome. Nature 1997, 387:151-158. 69. Massague J, Wotton D: Transcriptional control by the TGF-beta/Smad signaling system. EMBO J 2000, 19:1745-1754. 70. Li JM, Nichols MA, Chandrasekharan S, Xiong Y, Wang XF: Transforming growth factor beta activates the promoter of cyclindependent kinase inhibitor p15INK4B through an Sp1 consensus site. J Biol Chem 1995, 270:26750-26753. 71. Rich JN, Zhang M, Datto MB, Bigner DD, Wang XF: Transforming growth factor-beta-mediated p15(INK4B) induction and growth inhibition in astrocytes is SMAD3-dependent and a pathway prominently altered in human glioma cell lines. J Biol Chem 1999, 274:35053-35058. 72. Iavarone A, Massague J: Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-beta in cells lacking the CDK inhibitor p15. Nature 1997, 387:417-422. 73. Sherr CJ, Weber JD: The ARF/p53 pathway. Curr Opin Genet Dev, 10:94-99. 67.

78. Passegue E, Wagner EF: JunB suppresses cell proliferation by transcriptional activation of p16(INK4a) expression. EMBO J 2000, 19:2969-2979. This study establishes a positive role for JunB in transcriptional activation of p16 and provides a mechanism for cell-cycle arrest conferred by JunB. 79. Isoherranen K, Westermarck J, Kahari VM, Jansen C, Punnonen K: Differential regulation of the AP-1 family members by UV irradiation in vitro and in vivo. Cell Signal 1998, 10:191-195. 80. Pavey S, Conroy S, Russell T, Gabrielli B: Ultraviolet radiation induces p16CDKN2A expression in human skin. Cancer Res 1999, 59:4185-4189. Shows that p16 is indeed induced by UV radiation in human skin, as predicted from [78] and [79]. 81. Hengst L, Reed SI: Translational control of p27Kip1 accumulation during the cell cycle. Science 1996, 271:1861-1864. 82. Millard SS, Yan JS, Nguyen H, Pagano M, Kiyokawa H, Koff A: Enhanced ribosomal association of p27(Kip1) mRNA is a mechanism contributing to accumulation during growth arrest. J Biol Chem 1997, 272:7093-7098. 83. Zhang Y, Lin SC: Molecular characterization of the cyclindependent kinase inhibitor p27 promoter. Biochim Biophys Acta 1997, 1353:307-317. 84. Minami S, Ohtani-Fujita N, Igata E, Tamaki T, Sakai T: Molecular cloning and characterization of the human p27Kip1 gene promoter. FEBS Lett 1997, 411:1-6. 85. Ito E, Iwahashi Y, Yanagisawa Y, Suzuki Y, Sugano S, Yuasa Y, Maruyama K: Two short sequences have positive effects on the human p27Kip1 gene transcription. Gene 1999, 228:93-100. 86. Inoue T, Kamiyama J, Sakai T: Sp1 and NF-Y synergistically mediate the effect of vitamin D(3) in the p27(Kip1) gene promoter that lacks vitamin D response elements. J Biol Chem 1999, 274:32309-32317. 87. Servant MJ, Coulombe P, Turgeon B, Meloche S: Differential regulation of p27(Kip1) expression by mitogenic and hypertrophic factors: Involvement of transcriptional and posttranscriptional mechanisms. J Cell Biol 2000, 148:543-556.

88. Carrano AC, Eytan E, Hershko A, Pagano M: SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1999, 1:193-199. This study shows that SCFSKP2 is the protein-ubiquitin ligase required for p27 ubiquitination. It provides a critical target for the F-box protein, Skp2. 89. Montagnoli A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, Pagano M: Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev 1999, 13:1181-1189. The authors show that p27 ubiquitination requires Cdk2 kinase activity and formation of a trimeric complex containing p27, Cdk2 and cyclin E. 90. Nguyen H, Gitig DM, Koff A: Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on Cdk2 activity and the proteasome. Mol Cell Biol 1999, 19:1190-1201. Consistent with [89], this shows that cell-free degradation of p27 requires Cdk2 activity. It also demonstrates requirement for the proteasome. 91. Lisztwan J, Marti A, Sutterluty H, Gstaiger M, Wirbelauer C, Krek W: Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway. EMBO J 1998, 17:368-383.

684

Cell multiplication

92. Thullberg M, Bartek J, Lukas J: Ubiquitin/proteasome-mediated degradation of p19INK4d determines its periodic expression during the cell cycle. Oncogene 2000, 19:2870-2876. This paper shows that p19 is an unstable protein, efficiently targeted by the ubiquitin/proteasome pathway. This, when coupled to periodic transcription of the p19 gene, explains the periodic accumulation of the protein. 93. Coller HA, Grandori C, Tamayo P, Colbert T, Lander ES, Eisenman RN, Golub TR: Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci USA 2000, 97:3260-3265. 94. Tsutsui T, Hesabi B, Moons DS, Pandolfi PP, Hansel KS, Koff A, Kiyokawa H: Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity. Mol Cell Biol 1999, 19:7011-7019. Cells from Cdk4 nullizygous mice are shown to be slow to enter the cell cycle from G0. Molecular analysis reveals an accumulation of inhibited cyclinECdk2p27 complexes. Presumably the lack of cyclin-DCdk4 complexes prevents CKI exchange that would normally occur as cells progress through G1. These results support the idea that a major function of D-type cyclins is the sequestration of Cip/Kip inhibitors away from cyclin-ECdk2 complexes. 95. Reynisdottir I, Massague J: The subcellular locations of p15(Ink4b) and p27(Kip1) coordinate their inhibitory interactions with cdk4 and cdk2. Genes Dev 1997, 11:492-503. 96. Jiang H, Chou HS, Zhu L: Requirement of cyclin E-Cdk2 inhibition in p16(INK4a)-mediated growth suppression. Mol Cell Biol 1998, 18:5284-5290. 97. Mitra J, Dai CY, Somasundaram K, El-Deiry WS, Satyamoorthy K, Herlyn M, Enders GH: Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a). Mol Cell Biol 1999, 19:3916-3928.

low levels of nuclear cyclin-DCdk complexes. Surprisingly, cell-cycle perturbation was minimal. 100. Vigo E, Muller H, Prosperini E, Hateboer G, Cartwright P, Moroni MC, Helin K: CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase. Mol Cell Biol 1999, 19:6379-6395. This study shows that that along with cyclin E, CDC25A is one of the critical E2F targets for S phase entry. It confirms that tyrosine dephosphorylation is essential for activation of G1 Cdks. 101. Blomberg I, Hoffmann I: Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol Cell Biol 1999, 19:6183-6194. Complementary to [100]. Shows that ectopic expression of CDC25A leads to advance of the G1/S transition, confirming that Cdk dephosphorylation is rate-limiting. 102. Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J, Lukas J: Rapid destruction of human Cdc25A in response to DNA damage. Science 2000, 288:1425-1429. The authors show that downregulation of CDC25A by ubiquitin-dependent proteolysis is an early response to DNA damage and that damage-mediated G1 arrest can be overridden by ectopic expression of CDC25A. This response precedes p53-dependent increase in p21 levels. 103. Jinno S, Hung SC, Okayama H: Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4. Oncogene 1999, 18:565-571. Cdk4 is shown to be a target of inhibition by tyrosine phosphorylation in the special context of cells treated with DNA damaging agents while emerging from quiescence. 104. Tiefenbrun N, Melamed D, Levy N, Resnitzky D, Hoffman I, Reed SI, Kimchi A: Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest. Mol Cell Biol 1996, 16:3934-3944. 105. Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE: Proteasomal turnoverof p21Cip1 does not require p21Cip1 ubiquitination. Mol Cell 2000, 5:403-410. This study suggests that the frequently observed correlation between protein ubiquitination and proteasomal degradation is insufficient to conclude that ubiquitination is a prerequisite for degradation. It remains to be determined how p21 is targeted to the proteasome without ubiquitination.

98. LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E: New functional activities for the p21 family of CDK inhibitors. Genes Dev 1997, 11:847-862. 99. Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, Sherr CJ: The p21(Cip1) and p27(Kip1) CDK inhibitors are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 1999, 18:1571-1583. Cip/Kip inhibitors have an in vivo role in assembly of cyclin-DCdk complexes. Fibroblasts from p21/p27 doubly nullizygous mice had extremely