Control of the Cell Cycle

Summary of Key Points

Most cells in postnatal tissues are quiescent. Exceptions include abundant cells of the hematopoietic system, skin, and gastrointestinal mucosa, as well as other minor progenitor populations in other tissues.
Many quiescent cells can reenter into the cell cycle with the appropriate stimuli, and the control of this process is essential for tissue homeostasis.
The key challenges for proliferating cells are to make an accurate copy of the 3 billion bases of DNA (S phase) and to segregate the duplicated chromosomes equally into daughter cells (mitosis).
Progression through the cell cycle is dependent on both extrinsic and intrinsic factors, such as growth factor or cytokine exposure, cell-to-cell contact, and metabolic constraints.
The internal cell cycle machinery is controlled largely by oscillating levels of cyclin proteins and by modulation of cyclin-dependent kinase (Cdk) activity. One way in which growth factors regulate cell cycle progression is by affecting the levels of the D-type cyclins, Cdk activity, and the function of the retinoblastoma protein.
Cell cycle checkpoints are surveillance mechanisms that link the rate of cell cycle transitions to the timely and accurate completion of prior dependent events; p53 is a checkpoint protein that induces cell cycle arrest, senescence, or death in response to cellular stress.
Checkpoints minimize replication and segregation of damaged DNA, or the abnormal segregation of chromosomes to daughter cells, thus protecting cells against genome instability.
Disruption of cell cycle controls is a hallmark of all malignant cells. Frequent tumor-associated alterations include aberrations in growth factor signaling pathways, dysregulation of the core cell cycle machinery, and/or disruption of cell cycle checkpoint controls.
Because cell cycle control is disrupted in virtually all tumor types, the cell cycle machinery provides multiple therapeutic opportunities.

Most cells in the adult body are quiescent—that is, they are biochemically and functionally active but do not divide to generate daughter cells. However, specific populations retain the ability to proliferate throughout the adult life span, which is essential for proper tissue homeostasis. For example, cells of the hematopoietic compartment and the gut have a high rate of turnover, and active proliferation is essential for the maintenance of these tissues. On average, about 2 trillion cell divisions occur in an adult human every 24 hours (about 25 million per second). The decision about whether to proliferate is tightly regulated. It is influenced by a variety of exogenous signals, including nutrients and growth factors, as well as inhibitory factors, and the interaction of the cell with its neighbors and with the underlying extracellular matrix. Each of these factors stimulates intracellular signaling pathways that can either promote or suppress proliferation. The cell integrates all of these signals, and if the balance is favorable, the cell will initiate a series of processes, collectively known as the cell cycle, that lead to cell division into two daughter cells.

During the past four decades, extensive effort has been placed on unraveling the basic molecular events that control the cell division cycle. Studies in a variety of organisms have identified evolutionarily conserved machinery that regulates eukaryotic cell cycle transitions through the action of key enzymes, including cyclin-dependent kinases (Cdks) and other kinases. It is essential that proliferating cells copy their genomes and segregate them to the daughter cells with high fidelity. Eukaryotic cells therefore have evolved a series of surveillance pathways, termed cell cycle checkpoints, that monitor for potential problems during the cell cycle process. Human cells are continuously exposed to external agents (e.g., reactive chemicals and ultraviolet light) and to internal agents (e.g., byproducts of normal intracellular metabolism, such as reactive oxygen intermediates) that can induce DNA damage. A major goal of specific cell cycle checkpoints is to detect DNA damage and activate cell cycle arrest and DNA repair mechanisms, thereby maintaining genomic integrity.

Anything that disrupts proper cell cycle progression can lead to either the reduction or the expansion of a particular cell population. It is now clear that such changes are a hallmark of tumor cells, which carry mutations that impair signaling pathways that suppress proliferation and/or activate pathways that promote proliferation. In addition, most (if not all) human tumor cells have mutations within key components of both the cell cycle machinery and checkpoint pathways. This characteristic has important clinical implications, because the presence of these defects can modulate cellular sensitivity to chemotherapeutic regimens that induce DNA damage or mitotic catastrophe. This chapter focuses on the mechanics of the cell cycle and checkpoint signaling pathways and discusses how this knowledge can lead to the efficient use of current anticancer therapies and to the development of novel agents.

Cell Division Cycle

Overview of the Cell Cycle Machinery

Cell division proceeds through a well-defined series of stages ( Fig. 4.1 ). First, the cell moves from the nonproliferative, quiescent (also known as G ₀ ) state into the first gap phase, or G ₁ , in which the cell essentially is readying itself for the cell division process. This process involves a dramatic upregulation of both transcriptional and translational programs, not only to yield the proteins required to regulate cell division but also to essentially double the complement of macromolecules so that one cell can give rise to two cells without a loss of cell size. Not surprisingly, this process takes a significant amount of time (from 8 to 30 hours in cultured cells) and energy. Studies with cultured cells show that mitogenic growth factors are essential for continued passage through the G ₁ phase. Specifically, if growth factors are withdrawn at any point during this phase, the cell will not divide. However, as it nears the end of the G ₁ phase, the cell passes through a key transition point, called the restriction point, whereupon it becomes growth factor independent and is fully committed to undergoing cell division. The cell then enters the DNA synthesis phase, or S phase, in which each of the chromosomes is replicated once and only once. This is followed by a second gap phase, called G ₂ , which lasts 3 to 5 hours, and the cell then initiates mitosis, or the M phase, a rapid phase (lasting about 1 hour) in which the chromosomes are segregated. On completion of mitosis, the daughter cells can enter quiescence or initiate a second round of cell division, depending on the milieu.

Figure 4.1, The cell division cycle. One round of cell division requires high-fidelity duplication of deoxyribonucleic acid during the S phase of the cell cycle and proper segregation of duplicated chromosomes during mitosis, or the M phase. Before and after the S phase and M phase, the cell transits through “gap” phases, termed G 1 and G 2 . Quiescent (G 0 ) cells require the appropriate mitogenic stimuli for reentry into the cell cycle by inducing D-type cyclins. These stimuli are required up to a specific moment, known as the restriction point, at which time the cell cycle becomes independent of the mitogenic signals. The cellular processes required for transition through the different cell cycle phases are controlled by the action of regulatory pathways and mostly are driven by the expression of specific cyclins and the activation of cyclin-dependent kinases. These transitions are monitored by signaling pathways known as the cell cycle checkpoints (represented by blue columns ) that function to arrest the cell cycle at different phases (G 1 /S, intra–S phase, G 2 /M) in the presence of DNA damage. In addition, the spindle assembly checkpoint (SAC) delays the exit from mitosis in the presence of defective chromosome alignment by inhibiting the degradation of cyclin B1 and the subsequent inactivation of cyclin-dependent kinases.

Progression throughout the different phases of the cell cycle depends on the activity of key molecules that drive transcription, translation, or the structural changes required for cell division ( Table 4.1 ). A large number of these changes are modulated by protein phosphorylation and dephosphorylation, but other molecular processes such as SUMOylation, acetylation, or ubiquitin-dependent protein degradation are crucial for ordered cell cycle progression. Many of these cell cycle regulators have been involved in tumor development or may be attractive targets for cancer therapy and will be introduced in the following sections.

Table 4.1

Representative Molecules Involved in Cell Cycle Regulation

Protein Family/Complex	Representative Members	Function
KINASES
Cyclin-dependent kinases	Heterodimeric complexes formed of a cyclin (A, B, D, and E types) and a Cdk (Cdk1, Cdk2, Cdk4, Cdk6); Cdk7 functions as a Cdk-activating kinase	Phosphorylation of multiple proteins to drive progression throughout the different phases of the cell cycle
Wee1/Myt1	Wee1, Myt1	Inactivation of Cdks
Aurora-A holoenzyme	Aurora-A and its nonkinase activator, Tpx2	Spindle dynamics, chromosome segregation, and cytokinesis
Chromosome passenger complex (CPC)	Aurora-B (Aurora-C?), Incenp, survivin, borealin	Chromosome segregation
Polo-like kinases	Plk1–Plk5	Centrosome function, chromosome segregation, and cytokinesis
NIMA-related kinases	Nek1–Nek11	Centrosome function and mitosis
Haspin	Haspin	Phosphorylates histone H3 to recruit the CPC
Mastl	Mastl	Inhibition of PP2A phosphatases
CDK INHIBITORS
INK4 proteins	p16 ^INK4a , p15 ^INK4b , p18 ^INK4c , p19 ^INK4d	Inhibition of Cdk4 and Cdk6 during G ₁ progression
Cip/Kip inhibitors	p21 ^Cip1 , p27 ^Kip1 , p57 ^Kip2	Cdk inhibition and other roles in transcription or the cytoskeleton
TRANSCRIPTIONAL CONTROL
Retinoblastoma family	pRB, p107, p130	Repression of the transcription of genes required for the cell cycle
E2F transcription factors	E2F1–E2F8	Transcription of genes encoding S-phase and mitotic regulators
PHOSPHATASES
Cdc14	Cdc14a, Cdc14b	Control of transcription and cell cycle progression
Cdc25	Cdc25a, Cdc25b, and Cdc25c	Cdk activation and cell cycle progression
PP1	Multiple complexes with different regulatory subunits	Protein dephosphorylation
PP2A	Multiple complexes with different regulatory subunits	Protein dephosphorylation; major Cdk-counteracting phosphatase
UBIQUITIN LIGASES
SCF	E3 ubiquitin ligase formed of Rbx1, Cul1, Skp1, and an F-box protein (e.g., Skp2 or βTrCP)	Targets multiple cell cycle regulators (e.g., p27 ^Kip1 or cyclin E) for ubiquitin-dependent degradation during interphase
APC/C	E3 ubiquitin ligase composed for multiple subunits including Cdc20 or Cdh1 as coactivator molecules	Targets multiple cell cycle regulators for ubiquitin-dependent degradation during mitosis (cyclin B, securin) and G ₁ (e.g., Aurora-A, Plk1, or Tpx2)
OTHER FUNCTIONS
Kinesins	More than 600 proteins including Eg5, CenpE, MCAK	Microtubule-based motor proteins that hydrolyze ATP to generate energy for movement along microtubule fibers
Cohesins and condensins	Smc family (1–4), Rad21, Pds5, and SA (Stag) proteins, among others	Structure and regulation of DNA
Kinetochore proteins	More than 100 proteins including the CENP (centromere-binding proteins) family, and the Knl1, Mis12, Ndc80, and Dam1 complexes, among many others	Linking the chromosomes to microtubules and regulation of microtubule dynamics
Mitotic checkpoint complex (MCC)	Mad2, Bub3, BubR1, and Cdc20	Inhibit the APC/C until complete bipolar attachment of chromosomes to the mitotic spindle

APC/C, Anaphase-promoting complex/cyclosome; ATP, adenosine triphosphate; Cdk, cyclin-dependent kinase; CPC , chromosomal passenger complex; NIMA, never in mitosis, gene A; SCF, Skp1–Cullin1–F-box.

Cyclin-Dependent Kinases and Their Regulators

The Cdks constitute a large subfamily of highly conserved Ser/Thr kinases that are defined by their dependence on a regulatory subunit, called a cyclin. The first identified human Cdk, called Cdk1 (originally cdc2), was cloned by virtue of its ability to complement a mutant cdc2 yeast strain. Subsequent studies identified additional human Cdks and determined that they regulate distinct cell cycle stages; for example, Cdk4 and Cdk6 regulate cell cycle entry, whereas Cdk2 may have specific roles during the G ₁ -to-S transition and S phase. Cdk1 is essential in the control of G ₂ and mitosis and also may play additional roles in earlier stages. The human genome encodes about 15 additional Cdks, although the functional relevance of many of them is still unknown.

The activity of these kinases is controlled by multiple regulatory mechanisms. Cdks act in association with a cyclin subunit that binds to the conserved PSTAIRE helix within the kinase. Cyclin binding causes a reorientation of residues within the active sites that is essential for kinase activity. The associated cyclin also determines the substrate specificity of the resulting cyclin/Cdk complex. The cyclins are quite divergent, especially in their N-terminal sequences, but they all share a highly conserved 100–amino acid sequence, called the cyclin box, that mediates Cdk binding and activation. As their name implies, cyclins originally were identified as proteins whose expression was restricted to a particular stage of the cell cycle because of cell cycle–dependent regulation of both cyclin gene transcription and protein degradation. The human genome encodes more than 25 cyclin-like proteins, yet only four distinct subclasses—D-, E-, A-, and B-type cyclins—are thought to play key roles in cell cycle regulation (see Fig. 4.1 ). Each of these classes has a few paralogs (e.g., cyclin D1, D2, and D3; cyclin E1 and E2; cyclin A1 and A2; and cyclin B1, B2, and B3). The relative roles of these paralogs are not completely clear in most cases. Although some functional redundancy may exist, published evidence suggests differences in regulation, expression pattern, and substrate specificity.

The activation of cyclin/Cdk complexes requires considerable posttranslational regulation. First, kinase activation is dependent on phosphorylation of a threonine residue that is adjacent to the active site (Thr160 in Cdk2). This phosphorylation is catalyzed by a kinase, called Cdk-activating kinase (CAK). In mammalian cells, phosphorylation occurs after cyclin binding. Although it appears that at least two mammalian CAKs exist, the major CAK is a trimolecular complex composed of Cdk7, cyclin H, and Mat1. The Cdk7/cyclinH/Mat1 complex also is required for the control of basal transcription via regulation of RNA polymerase II function. Second, when it is first formed, the cyclin/Cdk complex frequently is subject to inhibitory phosphorylation of Thr14 and Tyr15 residues within the Cdk's active site by the Wee1 (Tyr15) and Myt1 (Thr14 and Tyr15) kinases. Activation of the cyclin/Cdk complex is then dependent on the action of a dual-specificity phosphatase called Cdc25. Mammalian cells have three different Cdc25 proteins, called Cdc25a, Cdc25b, and Cdc25c, which show some specificity for different cyclin/Cdk complexes and cell cycle stages.

Cdks are modulated by a series of Cdk inhibitors (CKIs) that play a key role in restricting the activity of the cyclin/Cdk complexes in response to either external signals or internal stresses. The CKIs can be divided into two distinct families based on their biological properties. The first CKI family is named INK4, based on their roles as IN hibitors of Cd k 4. The INK4 family has four members called p16 ^INK4a , p15 ^INK4b , p18 ^INK4c , and p19 ^INK4d (encoded by the CDKN2A-D genes in humans). These INK4 proteins specifically prevent the binding of cyclins to monomeric Cdk4 and Cdk6 but do not inhibit other Cdks. The second CKI family is named Cip/Kip and includes three members: p21 ^Cip1 (also called p21 ^Waf1 ), p27 ^Kip1 , and p57 ^Kip2 (encoded by the CDKN1A-C genes in humans). These Cip/Kip proteins have two major activities. First, they do not bind to monomeric Cdks but associate with and inhibit the activity of cyclin/Cdk complexes already formed. Second, Cip/Kip proteins may promote the assembly of cyclin D/Cdk4/6 complexes without dramatically perturbing its kinase activity. This activity is modulated by phosphorylation of Cip/Kip proteins by Src, Jak2, and Akt kinases, directly linking Cdk regulation with the activity of these upstream mitogenic pathways. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration, and cytoskeletal dynamics.

Retinoblastoma Proteins and E2F Transcription Factors

The retinoblastoma protein (pRB) was originally identified by virtue of its association with hereditary retinoblastoma. It behaves as a classic tumor suppressor: affected persons inherit a germline mutation within one allele of the pRB-encoding gene, RB1 , and loss of heterozygosity is seen in all of the tumors. Subsequent studies showed that the transforming ability of small DNA tumor viruses, including human papillomavirus, adenovirus, and simian virus, was dependent on the ability of virally encoded oncoproteins (E7, E1A, and SV40, respectively) to bind and inhibit pRB. Moreover, the RB1 gene is inactivated in approximately one-third of all sporadic human tumors.

pRB and the pRB-related proteins p107 and p130, collectively known as the pocket proteins, are transcriptional repressors whose major function is to inhibit the expression of cell cycle–related proteins (see Table 4.1 ). This suppressive activity is largely dependent on the ability to prevent cell cycle entry through inhibition of the E2F transcription factors. The E2F proteins regulate the cell cycle–dependent transcription of numerous targets, including core components of the cell cycle control (e.g., cyclin E and cyclin A) and DNA replication (e.g., Cdc6, Cdt1, and the Mcm proteins) machineries. pRB regulates E2F through two distinct mechanisms. First, its association with E2F is sufficient to block the transcriptional activity of E2F. Second, the pRB/E2F complex can recruit histone deacetylases to the promoters of E2F-responsive genes and thereby actively repress their transcription. Cell cycle entry requires the phosphorylation of pRB by cyclin/Cdk complexes and the consequent dissociation of pRB from E2F.

Studies have identified eight E2F genes that encode nine different E2F proteins. Pocket proteins can regulate a subset of these factors: E2F1, E2F2, E2F3a, E2F3b, E2F4, and E2F5. These E2F proteins associate with a dimerization partner, called DP, and the resulting complexes function primarily as either activators (E2F1, E2F2, and E2F3a) or repressors (E2F4 and E2F5) of transcription under the direction of the pocket proteins. Observations have suggested that several of these factors may act either as positive or negative regulators of transcription, depending on the cell type or the differentiation state. Most classic E2F target genes are regulated by the coordinated action of these repressor and activator E2Fs.

Ubiquitin-Dependent Protein Degradation

The original observation that cyclin levels are tightly regulated during the cell cycle implies that these proteins are regulated not only at the transcriptional level but also at the protein level. It is now evident that ubiquitin-mediated protein degradation is a major regulatory mechanism to ensure ordered transition through the different phases of the cell division cycle. Ubiquitylation depends on an enzymatic cascade, in which ubiquitin ligases recruit specific substrates for modification. About 600 ubiquitin ligases are encoded by the human genome. Among them, the Skp1–Cullin1–F-box (SCF) and the anaphase-promoting complex/cyclosome (APC/C) are known for driving the degradation of cell cycle regulators to accomplish irreversible cell cycle transitions. SCF has three core components: a RING finger protein, called Rbx1, which recruits the E2-ubiquitin conjugate; a cullin (Cul1); and Skp1 ( Fig. 4.2 ). Skp1 acts to recruit a family of proteins, called F-box proteins, which determine the target specificity of the SCF complex. Once SCF binds its substrate, it transfers a ubiquitin molecule to lysine residues within the target protein to create a polyubiquitin chain, which targets the substrate to the proteasome for degradation.

Figure 4.2, Ubiquitin ligases. The Skp1–Cullin1–F-box (SCF) and anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligases play a key role in enabling forward passage through key cell cycle transitions. These ligases are both large complexes that include three core components: a scaffolding protein called a cullin in SCF, a protein that recruits the E2 and its associated ubiquitin molecule, and a specificity factor (called the F-box protein in SCF and the activator in APC/C) that recruits the substrate. SCF and APC/C catalyze polyubiquitination of their substrates, which acts as a signal for substrate degradation by the 26S proteasome. SCF has numerous substrates whose degradation promotes passage through the early stages of the cell cycle, including the cyclin-dependent kinase inhibitor p27 Kip1 , cyclin E, E2F-1, and Cdt1. APC/C is essential for mitotic progression (by promoting degradation of securin and the mitotic cyclins) and for cell cycle exit (by promoting degradation of multiple cell cycle regulators).

The APC/C is a much larger complex, but it also contains a RING finger protein, called Apc11, to recruit the E2-ubiquitin conjugate, and a core cullin subunit (Apc2). In addition, APC/C is activated by a cofactor that, in a manner comparable with that of the F-box proteins of SCF, establishes substrate specificity (see Fig. 4.2 ). Cdc20 is the mitotic cofactor of APC/C, and it targets several cell cycle regulators (A- and B-type cyclins, Nek2, and securin) during mitotic entry and the metaphase-to-anaphase transition. Cdh1 (also known as FZR1 in mammals) replaces Cdc20 during mitotic exit and is the cofactor responsible for the elimination of many cell cycle regulators in the G ₀ or G ₁ phase to prevent unscheduled DNA replication. Recent studies have provided new insights into the intricate relationship between ubiquitylation and the cell division apparatus, including new roles for atypical ubiquitin chains, new mechanisms of regulation, and extensive cross talk between ubiquitylation enzymes.

Mitotic Spindle and Mitotic Kinases and Kinesins

In addition to the central role of Cdks in the cell cycle, many other kinases play critical roles in cell cycle progression. Aurora and Polo kinases were first identified in genetic studies in flies as a result of their essential role in mitotic progression. Each of these families of kinases is represented by a single member in yeast, whereas three Aurora kinases (Aurora-A, Aurora-B, and Aurora-C) and five Polo-like kinases (Plk1 through Plk5) exist in humans (see Table 4.1 ). Aurora-A participates in several processes required for building a bipolar spindle, including centrosome separation and microtubule dynamics. Aurora-A is activated by Tpx2, and the Aurora-A–Tpx2 holoenzyme may have critical implications in tumor development. Aurora-B, on the other hand, is part of the chromosome passenger complex (CPC) that localizes to the kinetochores from prophase to metaphase and to the central spindle and midbody in cytokinesis. Other components of the CPC include INCENP, survivin, and borealin, and this complex regulates proper microtubule-kinetochore attachment and promotes biorientation during mitosis. Aurora-C may play similar roles to Aurora-B, although it is mostly expressed in germ cells and may play a specific role in meiosis and during the first embryonic cycles. Plk1 functions as a major regulator of centrosome maturation, mitotic entry, and cytokinesis, whereas Plk4 is a critical regulator of centriole duplication. The other members of the family, Plk2, Plk3, and Plk5, are mostly involved in stress responses during interphase or in neuron biology.

A different group of additional cell cycle kinases with potential interest in tumor biology is the never in mitosis, gene A (NIMA)–related kinase family (Nek1 through Nek11). Four of these proteins, Nek2, Nek6, Nek7, and Nek9, are involved in cell cycle progression, whereas all other family members are likely to play critical roles in cilia and centrioles. Nek2, the closest relative to the Aspergillus NIMA kinase, localizes to the centrosome and plays a role in establishing the bipolar spindle by initiating the separation of centrosomes and contributing to microtubule organization at the G ₂ /M transition by phosphorylating several centrosomal substrates. Nek2 also may play additional roles in chromosome condensation and the mitotic checkpoint. Nek9, Nek6, and Nek7 function in a kinase cascade that participates in centrosome separation and the formation and/or maintenance of the mitotic spindle.

A structurally different kinase, Haspin, is involved in the phosphorylation of histone H3 on threonine 3 (H3T3). This kinase is activated by Cdk1 and Plk1, and phosphorylation of H3T3 is required for proper Aurora-B localization and activity and coupling of mitotic chromosome structure with transcription.

The activity of all these kinases is functionally linked to many other proteins associated with chromosomes, microtubules, or different organelles, whose activity is essential for the structural changes associated with cell cycle progression and chromosome segregation. Among them, microtubule-associated proteins such as kinesin motor proteins determine the dynamic behavior of the mitotic spindle required for chromosome movement during mitosis. During mitosis, microtubules associate with chromosomes through a large protein assembly known as the kinetochore. The position of kinetochores in the centromeric region of chromosomes is determined by a complex epigenetic, DNA sequence–independent mechanism. Kinetochores regulate the proper bipolar attachment between microtubules and chromosomes in order to distribute the replicated genome from a mother cell to its daughters. Given the relevance of the kinetochore in genome integrity, the composition of the kinetochore and the identification of various physical and functional modules within its substructure is under deep investigation.

Cell Cycle Phosphatases

Cdc14 phosphatases are the major Cdk-counteracting proteins in yeast. Although two family members exist in mammals, their relevance in the cell cycle is not well understood. In eukaryotes, two major complexes, PP1 and PP2A, account for more than 90% of protein phosphatase activity. In fact, these enzymes correspond to hundreds of phosphatase complexes assembled from a few catalytic subunits (PP1α, PP1β/δ, and PP1γ1/2 for PP1, and the Cα and Cβ isoforms for PP2A) and a diverse array of regulatory subunits. Recent evidence suggests that these protein families cooperate in the dephosphorylation of most cell cycle kinase targets, including the retinoblastoma family or mitotic phosphoproteins. PP1 and PP2A are major phosphatases responsible for pRB dephosphorylation during mitotic exit, although the relative roles of these complexes or the particular holoenzymes involved are not clear. Similarly, both PP1 and PP2A are required for dephosphorylation of hundreds of mitotic proteins that are phosphorylated by Cdk1, as well as the other mitotic kinases. Thus it has been suggested that the cell cycle ultimately is regulated by the dynamic equilibrium between Cdks (and partially by the other mitotic kinases) and PP1/PP2A activity. In the absence of Cdk activity, the balance tilts in favor of the phosphatases. When Cdks are activated, phosphatase activity is overtaken.

Cdk1 is able to directly inhibit PP1 by direct phosphorylation of the catalytic subunit. The Cdk-dependent inhibition of PP2A, on the other hand, is not direct; rather, it is mediated by a new kinase known as Greatwall in flies and Xenopus or Mastl in mammals. Cdk1 phosphorylates and activates Mastl, which in turn phosphorylates Arpp-19 and Ensa, two highly related proteins that function as inhibitors of a particular PP2A holoenzyme encompassing a regulatory subunit of the B55 family. Reactivation of PP1 and PP2A phosphatases is a mandatory step for the exit from mitosis and the transition to interphase.

Entry Into the Cell Cycle

Because most adult cells are quiescent, the mechanisms that determine their quiescent state and their reentry into the cell cycle with the appropriate stimuli are key determinants of tissue homeostasis. In quiescent cells, several DP/E2F complexes associate with the promoters of E2F-responsive genes and recruit pRB-family members, along with their associated histone deacetylases, to actively repress their transcription. This repression machinery therefore prevents the expression of proteins required for DNA synthesis and chromosome segregation. In addition, CKIs normally are expressed in quiescent cells, preventing the activation of Cdks. D-type cyclins are present at very low levels in most quiescent cells, in large part because they are phosphorylated by an abundant kinase called Gsk3β and then exported to the cytoplasm for degradation.

The transcription of D-type cyclins is induced in response to a wide variety of mitogenic stimuli. In addition, Gsk3β is inhibited by mitogens, thus preventing the degradation of these cyclins. D-type cyclins then associate with Cdk4 and Cdk6, and the resulting complexes phosphorylate pRB proteins, partially inactivating their transcriptional suppressor function ( Fig. 4.3 ). pRB inactivation causes the release of its associated DP-E2Fs. Repressive E2Fs such as E2F4 and E2F5 dissociate from the DNA, and the free E2F complexes—DP-E2F1, DP-E2F2, and DP-E2F3—now occupy the promoters and activate their transcription. DP-E2F targets include many genes encoding essential proteins required for DNA synthesis as well as mitosis. Through the control of pRB proteins, cyclin D-Cdk4/6 activity is a central mediator of the reentry of cells into the cell cycle. Not surprisingly, Cdk4/6 activity is frequently hyperactivated in cancer cells, as described later. Recent data suggest that Cdk6 may have kinase-independent, transcriptional functions, although the exact mechanisms behind these functions and their relevance in tumor development deserve further investigation.

Figure 4.3, Entry into the cell cycle. The pocket proteins—pRB, p107, and p130—regulate a subset of the E2F family of proteins among many other transcription factors. The pocket proteins bind to these E2Fs during the G 0 /early G 1 phases, block their transcriptional activity, and recruit histone deacetylase (HDAC), which comprises repressive complexes. Mitogenic signaling leads to the induction of D-type cyclins and the activation of interphase cyclin-dependent kinase (Cdk) complexes, which phosphorylate the pocket proteins and release their associated E2Fs. This process allows the activating E2Fs to induce the transcription of genes required for DNA synthesis and mitosis.

Cyclin E is itself an E2F-responsive gene, and this regulation creates a strong feed-forward loop. Cyclin E binds to Cdk2, and this complex may promote further pRB inactivation. Cyclin E–Cdk2 also phosphorylates the CKI p27 ^Kip1 on Thr187. This action creates a high-affinity binding site for the SCF ubiquitin ligase bound to the F-box protein Skp2, leading to p27 ^Kip1 degradation during the G ₁ /S transition. Finally, cyclin E-Cdk2 phosphorylates itself on multiple sites, creating a recognition site for SCF-Fbw7/Cdc4 and thereby ensuring its own destruction. The fact that lack of Cdk2 in the mouse does not result in defective mitotic cycles suggests that the activity of this protein overlaps with other Cdks, with Cdk1 being the best candidate. Indeed, Cdk1 is able to bind interphase cyclins such as cyclin D and cyclin E, and it is sufficient for G ₁ /S transition, at least in the absence of other interphase Cdks. Cdk2 is however an essential role in meiosis, although the molecular basis for this requirement is not fully understood.

DNA Replication

The DNA replication machinery is optimized to ensure that the genome is copied once—and only once—in each cell cycle. This optimization is achieved through a two-step process that first establishes a prereplication complex (pre-RC) at each origin of replication, a process that is frequently referred to as origin licensing, and subsequently transforms pre-RCs into the preinitiation complex (pre-IC) that activates DNA replication ( Fig. 4.4 ). These two steps occur at distinct stages of the cell cycle to ensure that origins are licensed only once per cell cycle and rereplication cannot occur. Pre-RC formation takes place in the initial steps of the cell cycle. The first event in this process is the recruitment of the multiprotein complex called the origin recognition complex to the origin DNA. The origin recognition complex recruits additional proteins including Cdc6, Cdt1, and finally the mini chromosome maintenance (MCM) complex, a helicase that is required to unwind the DNA strands to form the pre-RC. Once cells enter S phase, the transformation of the pre-RC to the pre-IC requires the activity of two kinases: a Cdk and the Ddf4-dependent kinase, which is composed of the Dbf4 regulatory subunit and the Cdc7 kinase. The action of these kinases allows numerous additional proteins to associate with the pre-RC and form the pre-IC. Assembly of the pre-IC is thought to trigger DNA unwinding by the MCM complex, recruitment of the DNA polymerases, and initiation of the replication process, frequently called “origin firing.”

Figure 4.4, Origin licensing and firing. The origin replication complex (ORC) associates with replication origins. During the G 1 phase, Cdc6 and Cdt1 are loaded on chromatin, and they in turn load the mini chromosome maintenance (MCM) complex on chromatin, at which point licensing is considered complete, and the multiprotein complex is called the prereplication complex (pre-RC). Once cells pass the G 1 -to-S transition, this complex is activated to form the preinitiation complex (pre-IC), and DNA replication is initiated. Activation requires both cyclin-dependent kinase (Cdk) and Ddf4-dependent kinase (DDK) activity. It results in recruitment of numerous proteins and activation of the MCM complex, which unwinds the DNA. Subsequently, core components of the replication machinery, including DNA polymerase α and DNA polymerase ε, are recruited to initiation sites. The transition from pre-RC to pre-IC results in inhibition of Cdt1 by ubiquitin-mediated degradation and geminin binding. Origin licensing cannot occur again until activation of anaphase-promoting complex/cyclosome at the end of mitosis allows accumulation of Cdt1.

The transformation of the pre-RC to the pre-IC can occur at different time points in S phase, depending on whether the origin fires early or late. The system can tolerate this heterogeneity because the pre-RC is disassembled after firing and cannot reform until the subsequent cell cycle. This process occurs through several mechanisms. The MCM complex travels with the replication fork in its role as the DNA helicase. Some evidence also indicates that phosphorylation of Orc1 reduces its ability to bind to origins. Finally, and most important, Cdt1 is prevented from participating in pre-RC formation outside of the G ₁ phase in two distinct ways. First, Cdt1 is marked for destruction by ubiquitination. This process is mediated by SCF-Skp2 and particularly by an E4 ubiquitin ligase that includes Rbx1 (to recruit the E2-ubiquitin), a cullin (Cul4), Ddb1, and Dtl/Cdt2 (the substrate specificity factor). Important to note, this Cul4-Ddb1-Dtl/Cdt2 complex functions independently of Cdt1 phosphorylation. Instead, Cdt1 is targeted only when proliferative cell nuclear antigen is present on the DNA, which occurs primarily as a consequence of the initiation of DNA replication. Second, cells possess a protein called geminin that sequesters Cdt1 and prevents it from participating in pre-RC formation. Geminin is present specifically in S-, G ₂ -, and early M-phase cells. The APC/C ubiquitinates geminin and thereby triggers its destruction during mitotic exit. This action creates a window between late mitosis and the end of G ₁ phase (when APC/C-Cdh1 is inactivated) in which geminin is absent, and therefore Cdt1 is free to participate in pre-RC formation.

The A-type cyclins are first transcribed late during the G ₁ phase under the control of the E2F transcription factors in a similar manner to that of cyclin E. Cyclin A associates with both Cdk2 and Cdk1 and acts both during S-phase and G ₂ /M. At the start of S phase, cyclin A–Cdk2 enters the nucleus and is specifically localized at nuclear replication foci, where it is thought to be actively involved in the firing of replication origins. As was described previously, cyclin A–Cdk2 also is required to phosphorylate E2F-1 and mediate its degradation, which is required to prevent E2F1 from triggering apoptosis.

Given the redundancy between different cyclin and Cdk family members, the specific role of cyclin A in DNA synthesis is unclear. Studies in mouse models identified a partially overlapping role between E- and A-type cyclins in the control of DNA synthesis in a cell-type specific manner. Fibroblasts lacking both cyclin A1 and cyclin A2 are able to proceed to S phase, and this is abrogated if E-type cyclins are deleted. However, A-type cyclins are essential in hematopoietic stem cells, suggesting cell-type differences probably caused by the levels of expression of the encoding genes. Although these observations suggest overlapping functions for E- and A-type cyclins in the activation of Cdks, a new kinase-independent role as an RNA-binding protein has been recently proposed for cyclin A2. Cyclin A2 binds directly to the 3′ UTR of Mre11 mRNA in a Cdk-independent manner to promote its translation. Mre11 is a component of the MRN complex, composed of Mre11, Rad50, and Nbs1, which plays a central role in DSB repair and replication fork restart. Depletion of cyclin A2 results in a reduction in Mre11 levels and defective repair of replication errors. Thus cyclin A2 participates in DNA synthesis in a Cdk-dependent manner while reinforcing stabilization of the key machinery responsible for repairing DNA lesions caused by replication errors.

Mitosis

The mitotic machinery is optimized to ensure that the replicated chromosomes are faithfully segregated to the daughter cells. This segregation is achieved through the use of a specialized microtubule-based structure, the mitotic spindle, on which the original chromosomes and their newly replicated copies, called sister chromatids, align and then are partitioned to opposite poles of the cell. The mitotic spindle is a highly dynamic structure that is maintained by many protein families, including motor molecules and other microtubule-associated proteins. The appropriate side-by-side alignment of the sister chromatids, termed biorientation, is facilitated by the physical tethering of the sister chromatids to one another. This process, called cohesion, actually occurs in S phase in a manner that is coordinated with the replication process. Cohesin is mediated by four proteins that together make up the cohesin complex. Two of these proteins, Smc1 and Smc3, have a long coiled structure with a dimerization domain at one end that allows them to heterodimerize to form a V -like structure. Important to note, the remaining ends of Smc1 and Smc3 can associate with each another to form a functional adenosine triphosphate (ATP) domain. This domain acts in an ATP-dependent manner to recruit two additional proteins, Scc1 and Scc3, which form a closed-ring structure that most likely encircles the chromosomes. Cohesin loading onto chromosomes, catalyzed by a separate complex called kollerin, is thought to be mediated by the entry of DNA into cohesin rings, whereas dissociation, catalyzed by Wapl and several other cohesin subunits, is mediated by the subsequent exit of DNA. Increasing evidence indicates that cohesin participates in other cellular processes that involve DNA looping such as transcriptional regulation. Interesting to note, mutations in genes encoding cohesin subunits and other regulators of the complex have been identified in several tumor types.

The related family of chromosomal proteins, condensins (condensin I and condensin II), are formed from a conserved pair of Smc proteins (Smc2 and Smc4) and distinct sets of non-SMC regulatory subunits. Condensins participate in a diverse array of chromosomal functions including chromosomal organization in interphase or the assembly of mitotic chromosomes.

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