els || cell cycle: regulation by cyclins

Cell Cycle: Regulation byCyclinsAndrew W Truman, University of Chicago, Chicago, Illinois, USA

Ana A Kitazono, Universidad Agraria La Molina, Lima, Peru

Jonathan N Fitz Gerald, Rhodes College, Memphis, TN, USA

Stephen J Kron, University of Chicago, Chicago, Illinois, USA

The cell cycle is defined as the periodic occurrence of

events that result in chromosome duplication (deoxy-

ribonucleic acid, DNA replication in S phase) and separ-

ation (mitosis). This process is directly regulated by both

external stimuli (such as nutrient availability) and

internal stimuli (such as cell size and DNA integrity). These

events are co-ordinately driven by the cyclin-dependent

kinases (CDKs). Although the expression of CDKs typically

remains relatively constant, their activities are highly

regulated by CDK-binding proteins known as Cyclins.

Cyclins are structurally related proteins whose levels

fluctuate throughout the cell cycle. Cyclin levels in the cell

are dynamically regulated through tight control over

both their rate of synthesis and degradation via ubiquitin-

mediated proteolysis. These CDK activators also impart

distinct substrate specificity to CDKs for the temporal

regulation of cell division.

Introduction

The defining events of the cell division cycle are deoxy-ribonucleic acid (DNA) replication during synthesis (S)phase and chromosome separation during the mitotic (M)phase. Each phase occurs after a ‘gap’ period (G1 and G2,respectively), during which little, if any, cytological changeis observed. Curiosity about what is happening duringthese gap phases has led to a remarkable biochemicalunderstanding of the determinants of cell cycle pro-gression. One early theory, based upon observations ofmitotically active cell extracts, suggested that the accu-mulation of material during interphase preceded entrance

into mitosis. This provided the first clue towards theidentification of a biochemical entity governing cell cycleprogression. See also: Cell Cycle

What are Cyclins? Roles in the CellCycle and Historical Background

Early studies described the existence of a ‘maturation-promoting factor’ (MPF). This heat-labile, soluble, cyto-plasmic compound was capable of making frog oocytesprogress into meiosis from arrest in G2. Oocytes at thisstage arrest to increase their volume, and remain arresteduntil surrounding follicle cells secrete progesterone, initi-ating entrance into meiosis. They arrest again at meiosis IIuntil fertilisation occurs. The ability of MPF to inducemeiosis was tested by transferring cytoplasmic materialfrom hormonally induced meiotic oocytes into recipientarrested oocytes. The arrested cells proceeded to meiosisupon injection (Masui andMarkert, 1971). It was apparentthat dividing cells produced an activating compound thatcould propel quiescent cells into division. Butwhatwas thisproduct? See also: MeiosisIt was discovered that certain proteins accumulate

periodically during the eukaryotic cell cycle. Theywere firstidentified in sea urchin egg extracts by studying changes inthe levels of proteins throughout the cell cycle. The obser-vation that these proteins abruptly disappeared at the endof mitosis and subsequently reappeared during interphaseindicated a cell cycle function related to induction ofmitotic events, and led to their naming as ‘cyclins’. Similarexperiments in clam oocytes demonstrated the existence oftwo different cyclin proteins, suggesting that the functionsand regulatory mechanisms of cyclins were conserved. Theconcordance between the kinetics of activation of thealreadyknownMPF (later identified as the cyclinB–CDC2complex) and the time of accumulation of the discoveredcyclin was also recognised, and a probable ‘direct cor-respondence’ of both processes was envisaged (Evans et al.,1983). See also: Mitosis

Advanced article

Article Contents

. Introduction

. What are Cyclins? Roles in the Cell Cycle and Historical

Background

. 3D Structure of Cyclins, Cyclin-dependent Kinases and

Regulation of Cyclin-dependent Kinases during the Cell

Cycle

. Controlling Elements Required for Cyclin Destruction

. Cyclin Regulation by Molecular Chaperones

. Cyclins and Senescence

. Summary

Online posting date: 15th May 2012

eLS subject area: Cell Biology

How to cite:Truman, Andrew W; Kitazono, Ana A; Fitz Gerald, Jonathan N; and

Kron, Stephen J (May 2012) Cell Cycle: Regulation by Cyclins. In: eLS.John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0001364.pub3

eLS & 2012, John Wiley & Sons, Ltd. www.els.net 1

http://dx.doi.org/10.1002/9780470015902.a0001354.pub2


http://dx.doi.org/10.1038/npg.els.0001356

Thereafter, several experiments confirmed the essentialrole of cyclins in the mitotic transition. Injecting the mes-senger ribonucleic acid (mRNA) for clam cyclin A intoXenopus oocytes were found to induce their release fromthe G2/M arrest at the first meiosis (Swenson et al., 1986).Experiments studying the effect of depletion of cyclinmRNA levels in cell extracts led to the recognition thatcyclins work as specific activators of the onset of mitosis.Xenopus cell extracts depleted of cyclinmRNAwere unableto undergo mitosis unless RNAase inhibitors were addedalong with new cyclin mRNA. These experiments showedthat synthesis of cyclin proteinwas sufficient for themitoticevents to take place (Murray and Kirschner, 1989). Theidentification of cyclin B as the activator subunit of theMPF, and p34/CDC2 as the catalytic subunit, wasachieved in a relatively short but extremely productiveperiod, through a series of elegant experiments derivedfrom genetic and biochemical observations (Nurse, 1990).See also: Microinjection into Xenopus Oocytes

Parallel to the studies in frog oocytes, yeast geneticapproaches had identified many genes as prospectivemitotic inducers and regulators. Cloning and sequencing ofthe fission yeast (Schizosaccharomyces pombe) cdc2+ geneallowed its classification as a protein kinase, an excitingfinding that correlated well with the frequently observedphosphorylation pattern changes during the cell cycle. Bylooking for genes that could allow growth of a fission yeastcdc2 mutant, the cyclin-dependent kinases (CDKs) ofbudding yeast (Saccharomyces cerevisiae) and humanswere cloned. Later, antibodies directed against Cdc2 wereshown to cross-react with the 34 kDa subunit of MPF.Also, antibodies against cyclin B cross-reacted with aregulator of CDC2 in fission yeast (Cdc13). This finallyprovided the biochemical identity of the mitosis-inducingprotein complex in eukaryotic cells (Nurse, 1990). See also:Protein Kinases: Physiological Roles in Cell Signalling

Cyclins comprise a large anddiverse family of proteins of50–90 kDa that share weak homology in their amino acidsequence. Within the primary structure, the ‘cyclin box’ (aregion of approximately 100 amino acids important forinteraction with the respective kinase partner) shows thehighest conservation (Andrews and Measday, 1998).Transcriptional and post-transcriptional mechanisms areresponsible for the regulation of cyclin levels (Figure 1). Thefirst level of regulation is the transcription of cyclin genes.Cyclin mRNAs are produced by a finely tuned transcrip-tion machinery that is sensitive to transitions in the cellcycle. This process involves cell-cycle-specific transcriptionfactors and the activity of cyclin–CDK complexes them-selves. Once expressed, the cyclins are also regulated at thelevel of protein abundance. At the end of mitosis, mitoticcyclin proteins disappear abruptly. This is due to theircoupling with ubiquitin – a small protein that marks itstarget for rapid degradation. Localisation is anothermeansby which cyclin activating function is regulated. Mitoticcyclins, for example, localise to the cytoplasm duringinterphase and are actively transported to thenucleus at theG2/M transition. This process seems to involve the

presence of nuclear localisation and export signals andphosphorylation steps. Cyclin localisation may also beunder the regulation of the DNA damage checkpointcontrols, to ensure a delay in entry into mitosis untilcompletion of DNA repair. See also: Ubiquitin PathwayStudies in different organisms have confirmed the pre-

diction that there are different types of cyclins that conferdistinctive substrate specificity to the catalytic subunit ofthe cyclin–CDK complexes. The programmed phos-phorylation of specific substrates then drives the cellthrough the different phases of the cell cycle (Figure 2)(Miller and Cross, 2001). All cyclins share strong hom-ology at the cyclin box region (Table 1 and Table 2). Theimplication of this knowledge is vast because of the rolethat cyclins have in cell growth and oncogenesis. The fol-lowing sections describe inmoredetail important aspects ofcyclin structure, function and physiology.

3D Structure of Cyclins, Cyclin-dependent Kinases and Regulation ofCyclin-dependent Kinases during theCell Cycle

The amino acid sequences of cyclins show no strikinghomology, except for the region that corresponds to the‘cyclin-box fold’. The structure of a truncated version ofbovine cyclin A (equivalent to human cyclin A 171–432fragment) showed that the cyclin box, running from resi-dues 199 to 306, forms a compact domain of five a helices.Remarkably, the presence of another equivalent andsuperimposable domain at the C-terminus was recognisedduring these studies. This result was unexpected from the

G1 S G2 M

Mammalian

Yeast

Clb5,6

Clb1,2

Clb3,4Cln1,2Cln3

Cyclin DCyclin B

Cyclin ACyclin E

Figure 1 Cyclin expression and degradation in the yeast and mammalian

(human) cell cycle. The combination of both protein level and activity are

depicted in the figure. D cyclin expression is induced by mitogenic signals

and can be found in the cell during two successive cell cycles under the

constant presence of the signal. However, the main role of D cyclins is at

the G1/S transition.

Cell Cycle: Regulation by Cyclins

eLS & 2012, John Wiley & Sons, Ltd. www.els.net2




analysis of the primary structure, since no resemblance tothe N-terminus ‘cyclin box’ amino acid sequence had beenpreviously detected (amino acid sequence identity lessthan 13%). This finding prompted further analysis of

potentially related sequences and led to the identification ofother proteins that may also present similar cyclin struc-ture. Among these, regions of p107 (related to the retino-blastoma protein, pRB), TFIIB (transcription factor) andp35 (activator subunit of CDK5) were found to acquire thecyclin fold. This suggests that the ‘cyclin-box fold’may be adomain that mediates protein interactions related to CDKregulation and transcription (Figure 3).CDKs play a key role in driving the cell cycle, and

regulation of their activation or inhibition is under thecontrol of different elements in a finely tunedprocess. CDKproteins are inactive unless they are bound to theirrespective cyclin partners. Upon binding, each cyclin–CDK complex is further subjected to negative and positiveregulation through specific phosphorylations. Phos-phorylation at a threonine residue near the C-terminus(Thr160 for CDK2) by the CDK-activating kinase (CAK)is necessary for activity. On the other hand, phosphoryl-ation of two residues near the N-terminus (Thr14 andTyr15) is inhibitory. Further negative regulation is pro-vided by the binding of specific CDK inhibitors (CKI).Mammalian CKIs are classified into two families, theCIP/KIP (CDK2/kinase inhibitor protein) and the INK4(inhibitors of cyclin-dependent kinase 4) inhibitors. Bothgroups restrain cell cycle progression by specificallyand coordinately binding to CDK complexes controllingG1 and G1/S phases. The CIP/KIP family consists ofp21/CIP1, p27/KIP1 and p57/KIP2. These inhibitors areless specific, inhibitingCDK2,CDK4andCDK6activities.They are important for both p53- and transforming growthfactorb(TGFb)-dependent regulation of the cell cycle. TheINK4 inhibitors are p16/INK4A, p15/INK4B, p18/INK4C and p19/INK4D. These bind specifically to cyclinD–CDK4 and –CDK6 complexes. See also: Apoptosis:Molecular Mechanisms; Cell CycleSolving the structures of different forms of CDK2 and

cyclin A has revealed shared principles of the mechanismsbehind the action, activation and inhibition of CDKs. Thestructure ofCDK2 is formed by two domains, and stronglyresembles that of the cyclic adenosine monophosphate(cAMP)-dependent protein kinase catalytic subunit. TheN-terminal domain is formed by the first 85 amino acidresidues and is rich in b-sheet structures. The C-terminaldomain consists of a much larger, a-helical lobe. A deepcleft between the two lobes defines an active site, where theresidues that bind adenosine triphosphate (ATP) and thoseresponsible for catalysis are located. Since all knownCDKsshare high homology, ranging from 55 to 65%, these allmay also present similar topology, explaining the goodconservation of the regulatorymechanisms identified so far(Jeffrey et al., 1995).In the unbound state, CDK2 is inactive for two reasons.

The first is the presence of a flexible loop from the C-terminal domain (the T loop, which includes Thr160, thetarget phosphorylation site of CAK) blocking entrance tothe active-site cleft. The second is the incorrect positioningin thewell-conserved ‘PSTAIRE’ (Pro–Ser–Thr–Ala–Ile–Arg–Glu) helix of residues that are important for ATP

APC

Proteasome

M

I

Key:

Cyclin B–Cdk1 (inactive)

Cyclin B–Cdk1 (active)

Cyclin B

Cdk1 (inactive)

Ubiquitin

Figure 2 Cyclin B and Cdk1 protein modifications during M and

interphase (I). Cdk1 is phosphorylated after binding with cyclin B and the

complex is fully activated by dephosphorylation at the Thr14 and Tyr15

residues, resulting in the onset of mitosis. During anaphase, cyclin B is

targeted for proteasome-dependent degradation by the APC, an ubiquitin-

protein ligating enzyme.

Table 1 Human cyclins

Cyclin CDK partner Function

Cyclin A1, A2 Cdk2, Cdk1/

Cdc2

S phase and G2

Cyclin B1, B2, B3 Cdk1 M phase

Cyclin D1 Cdk4, Cdk6 G1



Cyclin E1, E2 Cdk2 G1/S

Cyclin C Cdk8 Transcription

Cyclin F Cdk1 G2, M phase,

localization of

cyclin B1

Cyclin G Cdk5 p53-responsive,

PP2A

recruitment

factor for Mdm2

Cyclin H Cdk7 CDK-activating

kinase (CAK),

transcription

Cyclin I Spermatogenesis

Cyclin K Transcription

Cyclin L PITSLRE Pre-mRNA

splicing

Cyclin T1, T2 Cdk9 Transcription






binding and transfer. The determination of the crystalstructure of the cyclinA–CDK2 complex shed light onhowcyclin binding results in the activation of the catalyticsubunit. Attachment of the cyclin subunit enforces a

shearing between theN-terminal and C-terminal domains.This exposes the catalytic residues and regions importantfor substrate binding. This transition also effectivelyreconstructs the active site by bringing catalytic residues

Table 2 Yeast Cdc28 cyclins

Cyclin Expression Function Substrates

Cln1 G1 Start Far1, Sic1, Cdc3, Stb1,

Inhibits Clbs proteolysis

Activates Sic1 proteolysis

Inhibits pheromone response

Activates cytoskeleton polarization

Cln2 G1 Start Far1, Sic1, Lte1, Cdc3, Stb1, Ste20, Whi5

Inhibits Clbs proteolysis

Activates Sic1 proteolysis

Inhibits pheromone response

Activates cytoskeleton polarization

Inactivates Whi5, resulting in derepression of

G1/S transcription

Cln3 M phase/G1 Start Far1, Whi5

Inactivates Whi5, resulting in derepression of

G1/S transcription

Cln1, Cln2 transcription

Clb1 G2 Bud growth in G2 Swi5?

Mitosis

Nuclear division

Clb2 G2/M phase Bud growth in G2 Swi5, Cdc6, Swi4 Lte1, Pds1, Cdc16, Cdc23,

Cdc27, Swi6, Ndd1, Cdh1Mitosis

Nuclear division

Clb3 G2/M Mitosis Swi5?, Ndd1

Mitotic spindle formation

Clb4 G2/M Mitosis Swi5?, Kar9

Mitotic spindle formation

Clb5 S phase DNA replication Cdh1, Cdc6, Sld2, Swi5?, ORC?, MCM?

Mitotic spindle orientation

Clb6 S phase DNA replication Sld2, Swi5?, Cdc6?, ORC?, MCM?

Cyclin A Retinoblastoma protein TFIIB

Figure 3 Conservation of the ‘cyclin-box fold’ among cyclin A, the retinoblastoma protein and the transcription factor TFIIB.



into their appropriate conformation (Figure 4). Interest-ingly, no significant change in the structure of cyclin A isobserved uponbinding toCDK2.This suggests that cyclinsmay present in general a rather rigid structure that forcesspecific structural changes in the respective CDK partner.In the cyclin–CDK pair, it is the unstable, cell-cycle-regu-lated cyclin moiety that sets up the final three-dimensional(3D) conformation of the catalytic structure, determiningnot only substrate specificity but also interactionwithotherregulatory factors (Figure 4) (Morgan, 1997).

Similarly, pivotal information was obtained by solvingthe structure of the cyclin A–CDK2 complex bound to afragment of the CKI p27/KIP1 containing the minimumregion required for inhibition. The fragment was found tobind to both subunits. A short peptide containing thesequence Leu–Phe–Gly from p27 interacts with a bindingpocket in cyclin A. More importantly, binding to the N-terminal domain of CDK2 results in a disruption of itsstructure and in blocking of ATP binding.

INK4 inhibitors are characterised by the presence ofseveral 30-amino acid motifs known as the ankyrin repeats(four in p16 and five in p19) that stack together giving theprotein an ‘L’ shape. The crystal structure of CDK6 boundto either p16/INK4A or p19/INK4D suggests that themechanisms used by the Ink4 inhibitors differ in severalaspects from those used by the Cip/Kip inhibitors. Bothp16 and p19 bind to one side of the catalytic cleft of CDK6,opposite to the cyclin-binding site. The interaction affectsboth domains of CDK6 and results in a twist that distortsthe cyclin- andATP-binding sites. This impedes the shift ofthe PSTAIRE helix into the active site, which is induced bycyclin binding and is required for activity.

D Cyclins

Three types of mammalian D cyclins have been identified:D1, D2 and D3. These show approximately 60% amino

acid sequence identity. Although the expression of eachseems to be cell-type-specific, D3 and eitherD1 orD2 typesare found in most cells. D cyclins act early in the cell cycle(Figure 1), activating CDK4 and CDK6, and committingthe cell to one round of DNA replication and mitotic div-ision, establishing the cellular ‘restriction point’. Growthfactors have a direct effect on cyclin D expression, a keystep in forcing the cell to reenter the cell cycle, from thequiescent G0 to the active G1 phase. In general, theexpression pattern of cyclins is cell-cycle-regulated, but theD cyclins are unique in that they respond to externalmitogenic signals, functioning as cell division initiators.Further, an important noncatalytic role for cyclin D–CDK4 complexes is the sequestration of the p27/KIP andp21/CIP inhibitors, which promotes the activation ofcyclin E–CDK2 and progression into S phase. Since cellproliferation can be induced by upregulating levels ofcyclin D, these cyclins are particularly prone to act asoncogenes. See also: OncogenesCyclin D1 was identified among other cyclins in a screen

for human genes that were able to rescue a yeast straindeficient in its G1 cyclins. Another screen for genes thatwere inducible by cytokines also identified cyclin D1.Among the cyclins, mutations resulting in upregulation ofD cyclins are found most frequently in cancer cell lines.Cyclin D1, for example, localises on chromosome 11q13, aregion linked to several proliferative disorders, and wasoriginally isolated as an oncogene formed by chromosomaltranslocation in parathyroid adenoma cells. Althoughthere are still many unknown features of the role of cyclinD1 in proliferative control, its participation in carcino-genesis seems to be certain. For example, many tumourtypes exhibit higher cyclinD1 protein levels (particularly inlung, pancreatic, breast and prostate cancers). Thisincrease in expression arises through differentmechanisms.Mutations in the 3’untranslated regionof theCCND1generesulting in stabilisation of CCND1 mRNA have been

CDK2 CDK2-Cyclin A INK4-CDK2p27-CDK2-Cyclin A

Figure 4 Structural transformations in CDK during activation and inhibition. CDKs (blue), by themselves, are inactive. Activation occurs through

phosphorylation of the T loop (green) and the binding of cyclin (purple) at the PSTAIRE helix (red). These events lead to a conformational change that

produces a functional active site (yellow, see text for details). Tertiary inhibitors of the CIP/KIP1 family (p27; orange) block kinase activity by disrupting the

N-terminal domain and penetrating into the catalytic site. Binary inhibitors of the INK family (p19; salmon) distort the CDK N-terminal domain.




reported in mast cell leukaemia. Stabilising mutations anddeletions around Thr 286 (required for cyclin degradation,see section ‘Controlling elements for cyclin destruction’)have been detected in endometrial and throat cancers.Another way cyclin D1 can become overexpressed isthrough the constitutive activation of mitogenic signallingpathways, such as the ERK1/2 pathways that is often seenin oncogenesis.

Further, the retinoblastoma protein (pRB), a knowntumour suppressor, has been identified as a substrate ofcyclin D–CDK complexes. pRB is a negative regulator ofG1 progression that exerts its function by downregulatingE2F, a transcription factor that promotes DNA repli-cation. Phosphorylation of pRB releases E2F from theinhibitory activity, and the E2F-dependent transcriptionof genes is then actively driven promoting progression intoS phase. See also: Tumour Suppressor Genes

Interestingly, besides its role as CDK activator, cyclinD1 has also been found to interact directly with the oes-trogen receptor, causing upregulation of the receptor-mediated transcription and stimulation of proliferation.The interaction occurs at the hormone-binding domain ofthe receptor, and seems to be synergistic with oestradiolbinding. However, cyclin D1 alone is sufficient to upregu-late the oestrogen receptor’s transcriptional activatingcapabilities, making breast epithelial cell proliferationcontrol independent of hormone stimulation (Ciemerychet al., 2002). Accordingly, mammary gland cells are par-ticularly prone to oncogenic transformation when cyclinD1 levels are higher due to overexpression or amplificationof the gene. See also: Breast Cancer

The knowledge that overexpression of cyclins in cancermay contribute to cellular resistance to anticancer treat-ments have prompted studies on ways to inhibit cyclins.One promising technique involves the knockdown ofUSP2, a deubiquitylating enzyme that specifically targetscyclin D1. This treatment reduced the proliferation ofcancer cells (in which cyclin D1 is overexpressed), but notnormal fibroblasts (Shan et al., 2009).

Comparatively little is known regarding the otherD cyclins, D2 and D3, whose isolation by cross-hybrid-ization was mainly due to their high homology withcyclin D1. Cyclin D2 localises to chromosome 12p13.Its overexpression has been linked to the developmentof leukaemia in mice, and to testicular and colorectalcarcinomas. The important role of D cyclins in cell pro-liferation has been demonstrated by the phenotypes pre-sented by the D1 and D2 knockout mice. Mice lacking D1cyclins show defects in mammary gland and retinal devel-opment, and female mice lacking D2 cyclins show ovarianfailure that results in infertility, suggesting that theirexpression and function are tissue-specific. Studies onmiceexpressing a singleD-type cyclin have revealed that in thesemutants, the tissue specificity is lost at the embryonic stageand the cyclin is expressed ubiquitously (Ciemerych et al.,2002). At later stages, each mutant exhibits particularabnormalities that affect their long-term survival (mega-loblastic anaemia, neurological abnormalities and rudi-

mentary cerebella in mice expressing only cyclins D1, D2and D3, respectively).

Cyclin E

Cyclin E binds CDK2 and peaks in late G1. This complexalso phosphorylates pRB and since cyclin E expression isactivated by E2F, a positive feedback mechanism ensureshigh kinase levels for the onset of S phase. Cyclin E-dependent phosphorylation of pRB follows that by cyclinD kinase, resulting in two successive ways to release E2F.One is activated by mitogenic factors (growth factorinduction of cyclin D) and the other by intracellularmechanisms (cell cycle regulation of cyclin E). A differencearises, however, when pRB is deleted from the cell: whilecyclin D1 becomes dispensable for cell cycle progression,cyclin E does not, suggesting that the latter has a broadersubstrate specificity.CyclinEhas long been assumed to be the critical initiator

of DNA replication, directing CDK2 to phosphorylate theproteins that regulate unwinding of chromosomal originsof replication and activate DNA polymerases to performchromosomal replication. After the onset of S phase, cyclinE is phosphorylated by CDK2, targeted for degradationand replaced by cyclin A to drive cells through S phase. Adetailed description of the process by which theD, E andAcyclins organise the onset and progression of S phase is stilllacking. However, recent studies in mice where cyclins E1and E2 were disrupted, have revealed that their ‘critical’role in DNA replication needs to be reevaluated. Lack ofboth cyclins result in lethality but from causes unrelated tocell proliferation. These studies demonstrate that cyclin Ehas an essential function in cells undergoing progressionfrom G0 to S and endoreplication (placental trophoblastsand megakaryocytes), but not in embryonic cell cycles(Geng et al., 2003). Interestingly, CDK2 knockout micewere found to be viable but sterile, suggesting thatCDK2 isalso not required for embryonic development or continu-ous cell cycle progression, but is essential for germ celldevelopment and meiosis.

Cyclins A and B

These mitotic cyclins were among those initially identifiedin sea urchins and clams. It was their oscillating level thatgave the initial hints for a cell cycle function. Interestingly,cyclin A binds to both CDK2 and CDK1 functioning in Sphase and mitosis. The cyclin A–CDK2 complex acts ondifferent substrates such as DP-1 (a subunit of the E2Fcomplex) and CDC6 (a key regulator of DNA replication),thereby promoting progression into and through S phase.Cyclin A expression peaks during S phase. Cyclin AmRNA or protein depletion from G1 cells results ininhibition of DNA replication, and overexpression causesa premature entry into S phase. CyclinA binds to CDK2 assoon as cyclin E levels decrease, further promoting DNAreplication but concurrently preventing assembly of newreplication complexes. Two A-type cyclin genes have been





identified that exhibit particular functions and expressionpatterns. Although deletion of cyclin A2 results in embry-onic lethality, cyclin A1 knockout mice are viable butexhibit meiotic defects that result in male sterility. See also:Mitosis

Much is known about cyclin B, which shows the highestconservation not only in terms of CDKactivation, but alsoin the regulation of their degradation (see below). CyclinB–CDK1/CDC2 promotes most mitotic events fromassembly of the mitotic spindle to chromosome conden-sation and nuclear envelope breakdown. It also activatesthe molecular motors by which chromosomes are pulledfrom the metaphase plate and deposited into the daughtercells. The reforming of the nuclear envelope, chromosomedecondensation, spindle disassembly and cytokinesis nor-mally follow promptly after anaphase, and cannot proceeduntil the levels of cyclin B are greatly diminished.Accordingly, destruction of cyclin B (inactivation ofCDK1) is essential for exit from mitosis. Three B-typecyclin genes have been identified, which exhibit differentfunction and expressionpatterns. CyclinB2 knockoutmiceare viable with no obvious phenotype, whereas disruptionof cyclin B1 results in embryonic lethality (Brandeis et al.,1998). Cyclin B3 functions in meiosis and is expressedmainly in germ cells.

Cyclin C

Original observations that expression of cyclin C in yeastcan rescue lack of G1 cyclins and that expression of CyclinC is highest early inG1 suggested a role in the regulation ofG1 transcription. Subsequently, it has been shown thatCDK3 forms a complex with cyclin C that phosphorylatesRb during theG0/G1 transition, a process where resting orterminally differentiated cells re-enter the cell cycle (mim-icked in this case by depriving cells of growth serum forthree days, then stimulating cells with serum) (Ren andRollins, 2004).

Cyclin C has also been shown to bind CDK8, a CDKstructurally related to the yeast Srb10 kinase. Cyclin C-CDK8 forms a subcomplex with the Mediator proteinsMED 12 and MED13 that phosphorylates the C-terminaldomain of RNA polymerase II. This disrupts Mediator–Pol II interactions, allowing fine control of the transcriptlevels of an activated gene.

Cyclin F

Cyclin F is an essential for cell viability, but appears to bean ‘orphan’ cyclin in that is does not bind to or activate theactivity of any CDKs. The level of cyclin F oscillatesthroughout the cell cycle with maximum expressionoccurring at G2 in a similar manner to cyclin A and B.Unlike the other cyclins, proteolysis of cyclin F is likely toutilise metaloproteases. During G2, cyclin F forms acomplex with the SCG ubiquitin ligase and binds to cen-trioles, catalysing degradationofCP110, a protein essentialfor centrosome duplication. In this manner, cyclin F

contributes to genome integrity and correct progression ofmitosis (D’Angiolella et al., 2010).

Cyclin G

Much less is known about Cyclin G except that it is a p53-responsive gene and appears to have roles in trafficking(such as lysosomal enzyme sorting and receptor trafficking)(Susa et al., 2010). It does this by promoting clathrinrecycling from endocytic vesicles. Consequently, cellslacking cyclin G display decreased microtubule generationat kinetochores/chromosomes because of decreased cla-thrin in the mitotic spindle (Tanenbaum et al., 2010).

Cyclin K

Cyclin K forms a complex with CDK9 that binds tochromatin during the replication of DNA. Analysis of thischromatin associated complex shows CDK9 bound toataxia telangiectasiamutated (ATM) and other checkpointsignalling proteins, implying a direct role of Cyclin K–CDK9 inmaintaining genome integrity during S phase (Yuand Cortez, 2011).

Other cyclins

Since the current definition of a cyclin requires the presenceof the cyclin box domain and interaction with a CDK,several other cyclins have been described with transcrip-tional or unknown function. Mammalian cyclin H is theregulatory subunit of the major CDK-activating kinase(CAK), CDK7. CAK-dependent phosphorylation of aconserved threonine residue (Thr160 in Cdk2) is requiredfor the complete activation of CDKs. CDK7 shows highsequence homology to its substrate CDKs, whereas cyclinH shows only 15% identity to cyclin A in the usually highlyconserved cyclin box domain. The structure of cyclinH hasbeen solved, showing that despite the low homology, theoverall topology significantly resembles that of cyclin A.Other cyclins that have been identified in human cellsinclude L and T that regulate transcription.

Substrate recognition motifs in cyclins

Recent work has suggested an alternate theory of CDKactivity regulation: that CDK activity is not activated in apurely quantitave manner throughout the cell cycle, but isalso controlled by the specificity of the CDK-cyclin com-plex present at any given stage. For example, while it is truethat activity of Cdk1 increases through the cell cycletowards the traditional CDK1 consensus motif (S/T-P-X-R/K), purified Cln2–Cdk1 complex displays a distinctsubstrate specificity compared to that of Cln2–Cdk1complexes (Koivomagi et al., 2011).Several motifs have been identified in cyclins that are

essential for substrate binding and recognition. The firstidentified was the MRAIL motif present in a hydrophobicpatch on the surface of cyclin A, which serves as a dockingsite for substrates containing the RxL motif (Cy binding




motif (Wohlschlegel et al., 2001). Substrates that aretypically phosphorylated in the G1 and S cell cyclestages containmore RxLmotif repeats, consistent with theconcept that increased CDK–substrate interaction earlyin the cell cycle compensates for the low intrinsic activityof CDK–G1/S cyclins. The MRAIL motif resides at theN-terminal lobe within the first cyclin-box fold. A secondmotif, identified by the sequence FLRRxSK, is locatedat the second cyclin-box fold of cyclin B. Both motifsare located at opposite sides of the catalytic cleft inthe cyclin A–CDK structure, and are thought tocooperatively provide enhanced and specific intera-ction with substrates (Goda et al., 2003). Similarly, theL/VxCxE motif was identified at the N-termini ofcyclinsD andE as essential for binding to pRb (Kelly et al.,1998).

Controlling Elements Required forCyclin Destruction

Given the activating function of cyclins, themost rapid andeffective way to ensure their complete elimination is byproteolytic degradation. A key player in this process isubiquitin, a small polypeptide of 76 amino acids. Additionof a polyubiquitin chain to a lysine residue of a protein canmark it for degradation by the proteasome complex. Inthis system, the presence of certain ‘signatures’ such asthe ‘destruction box’ (‘D box’, consensus sequenceRxxLxxxxN) and phosphorylation at specific residuesdetermines the fate of the prospective substrate protein.See also: Ubiquitin Pathway

Proteasomes are ‘self-compartmentalised’ proteasesthat are structurally and mechanistically highly conse-rved in archaea, bacteria and eukaryotes. The eukaryoticproteasome is a 26S elongated structure, formed by acentral 20S core that harbours the catalytic sites, andmay be capped at either one or both ends by 19Scomplexes. Subunits located in these capping structuresseem to recognise and bind ubiquitinylated substrates,directing their entry into the 20S core for degradation.Multiple proteolytic activities lead to a nonspecific deg-radation of the proteins into peptides of around 7–9 resi-dues. Proteasome degradation is not exclusively directedtoward ubiquitinylated proteins; other substrates includeunfolded or misfolded proteins, antigens and others thatare targeted by different mechanisms. See also: ProteaseComplexes

Ubiquitinylation requires the participation of at leasttwo enzymes: E1, the ubiquitin-activating enzyme, whichbinds ubiquitin through a high-energy thiolester bond in astep that requires ATP hydrolysis; and E2, the ubiquitin-conjugating enzyme. The latter receives the ubiquitinmoiety from E1 and transfers it directly to a lysine residuein the substrate; more commonly, this last step is catalysedby a ubiquitin protein-ligating enzyme, E3. Eventually,several ubiquitin chainswould be added following the same

mechanism each time, resulting in the formation of iso-peptide bonds between the C-terminus of the newcomerubiquitin monomers and the e-amino group of lysine resi-dues in the last ubiquitin added.The cyclosome or anaphase-promoting complex (APC),

a 20S supramolecule, is the ubiquitin protein ligase(E3 enzyme) in charge of targeting mitotic cyclins andother substrates for degradation (Townsley and Ruder-man, 1998). Between 8 and 12 different proteins havebeen identified as components of the APC. The activityof the APC is under the control of the cyclin B–CDK1 complex, which causes its own inactivation andeventual exit frommitosis andprogression into the next cellcycle.Anaphase, characterised by sister chromatid segre-

gation, is driven by the APC-directed degradation ofelements such as the securins Pds1 (‘precocious dissoci-ation of sister chromatids’ protein (1) in S. cerevisiae) andCut2 (‘cell untimely torn’ protein (2) in S. pombe), whichact as ‘anaphase inhibitors’. This is followed by degrad-ation of cyclins at telophase, with the subsequent inacti-vation of the mitotic CDK and progression into interphase(G1).The coordinated progression through the nuclear cycle

requires the precise timed activation of APC, and also ahighly stringent substrate-discriminating activity. Thefindingof twodifferentAPCactivators seems to explain theways by which this coordination is accomplished. Cdc20(also known as Slp1/p55C/fizzy) is a conserved protein thatpromotes the APC-directed degradation of Pds1/Cut2.Later on a related proteinHct1 (also knownasCdh1/Srw1/fzr) directs APC for cyclin B degradation. Only Clb-CDKcomplexes can phosphorylate Cdc20, whereas both Cln-CDK Clb-CDK complexes can phosphorylate Cdh1. It isthought that this difference contributes to the prevention ofinappropriate cyclin degradation.Ase1 (a yeast microtubule-binding protein), Cdc5 (a

yeast polo-like kinase) and geminin (an inhibitor of DNAreplication) are other examples of proteins found to beAPC substrates at telophase.The proteolytic degradation of G1 cyclins is also

dependant on the 26S proteasome, but is directed by adifferent E3 complex, denoted as SCF in yeast (Skp1,Cdc53-, F -box). This activity requires the phosphorylationof specific residues within the C-terminal ‘PEST’ (Pro–Glu–Ser–Thr) region, at the consensus S/TP site for aCDK. Budding yeast Cln1, Cln2 and Cln3 degradation isdependant on the presence of several phosphorylation sitesand the Cdc34 E2 enzyme. The degradation of the yeastClb–Cdc28 kinase inhibitor, Sic1, is also dependant on thissystem. Interestingly, a similar mechanism seems to regu-late the mammalian inhibitor p27/KIP1 turnover. Hereagain, the substrate specificity seems to be given by thepresence of ‘adaptor proteins’ such as Cdc4 and Grr1,required for the degradation of Sic1 and G1 cyclins,respectively. Phosphorylation and the presence of thePEST motif also signal mammalian cyclin C, D and E fordegradation.






Cyclin Regulation by MolecularChaperones

Over recent years, research has uncovered an importantrole of molecular chaperones in cyclin function. The firstevidence came from the findings that deletion of the yeastHeat shock protein 70 (HSP70) co-chaperone moleculeYdj1 results in the delayed execution of START. Ydj1 wasshown to interact with the Cln3 G1 cyclin and over-expression of Cln3 suppresses the cell cycle defects seen incells lacking Ydj1. Cln3 contains a motif similar to a ‘J-domain’, known to be critical in mediating HSP70-cochaperone interactions. Seminal work demonstrated thatCln3 binds to yeast HSP70 through this domain in the G1cell cycle stage. The Cln3 J-domain-like motif is unusualsince it does not contain the HPDK repeat required toactivate the essentialATPase activity of the chaperone, andtherefore acts to inhibit molecular chaperone activity. InG1, the binding of Ydj1 to Ssa1 stimulates the ATPaseactivity of Ssa1 and results in the release of Cln3 fromSsa1.This allows nuclear accumulation of Cln3 to occur and theinitiation of G1/S transcription (Verges et al., 2007). In amanner parallel to the yeast model, Cyclin D1 associateswith the HSP70 isoform HSC70. HSC70 binds to newlysynthesised cyclin D1 and this interaction is required toform an active CDK4–Cyclin D1 complex. The moleculartrigger that regulates the association between cyclins andmolecular chaperones remains to be identified (Diehl et al.,2003). See also: Chaperones, Chaperonin and Heat-ShockProteins

Cyclins and Senescence

Whennormal cells are exposed toDNAdamage thatwouldmake a cell’s progeny nonviable, they may self-destruct viaapoptosis or enter a state known as senescence. In this statecells irreversibly exit the cell cycle (in contrast to differen-tiated cells that may re-enter the cell cycle) but are stillmetabolically active. It has become clear that this exiting ofthe cell cycle and therefore cyclins themselves are import-ant in achieving a state of cellular senescence.

Decreases in cyclinB1 levels resulting from the treatmentof cells with a sub-lethal level of the antibiotic Adriamycindirectly results in a large and flattened cell shape alongwithactivated b galactosidase activity, all hallmarks of cellularsenescence (Kikuchi et al., 2010).

CyclinD1appears tohave a significant role in promotingcellular senescence. It is expressed at significantly higherlevels in senescent cells. This increase is probably explainedby the finding that the 5’-untranslated region of the cyclinD1 gene contains an element that results in differentialexpression of cyclin D1 in young versus senescent fibro-blasts (Berardi et al., 2003). Cyclin D1may participate in apositive feedback cycle with p21, a protein known toaccumulate in senescence. p21 promotes nuclear accu-mulation of cyclin D1, which in turns stabilises p21.

Interestingly, senescent cells that express high levels ofcyclin D1 are impaired in the phosphorylation of Rb,because all cyclin D1–CDK complexes are associated withp21 (Dulic et al., 1993). See also: Cell Senescence In Vitro

Summary

Progression through the cell cycle requires the coordinatefunctioning of elements in charge of metabolic processes,cytoskeleton organisation, chromosome replication andsegregation, cell morphology, etc. Cyclins are ubiquitousregulatory subunits that bind and activate the CDKs,forming complexes that drive the eukaryotic cell cycle. Inthese complexes, the identity of the cyclin moiety mainlydetermines substrate specificity and, in some cases, sub-cellular localisation. Cyclin levels are controlled duringtranscription and via protein degradation, whereas thelevels of CDKs do not significantly change throughout thecell cycle. Different cyclin–CDK complexes are suc-cessively up- and downregulated by a series of inter-dependent phosphorylation reactions and also by thebinding of specific inhibitory and activating subunits. Themain cyclin–CDK complexes in human cells so far identi-fied are: cyclin D–CDK4, cyclin D–CDK6, cyclin E–CDK2, cyclin A–CDK2, cyclin A–CDK1, cyclin B–CDK1 and cyclin H–CDK7. Since the aberrant expressionof several of these elements results in oncogenesis, learningthe details of how cyclin–CDK complexes work and areregulated will have an immediate impact on cancer therapyand prevention.

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