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Cyclins are essential activating subunits of cyclin-dependent kinases (CDKs) and comprise a large familyof proteins conserved in function from yeast to humans.Cyclins originally came to our attention as cell-cycleregulatory proteins, but we now know that cyclin–CDKcomplexes have functions beyond cell-cycle control. Thebroad application of the cyclin–CDK model might reflectthe multiple levels of regulation that control the activityof CDK complexes, making them ideally suited torespond to multiple inputs. The multifaceted regulation ofcyclins and their CDK partners has been amply reviewedand we do not provide a detailed description of cyclin–CDK regulation here (reviewed in Refs 1–4). Rather, thecompletion of the yeast genome sequence provides anopportunity to survey recognizable cyclins and assesswhat we have learned about their function throughgenetic and biochemical analysis. We focus our review onthe functions of the various cyclins of budding yeast asrevealed by the phenotypes of cyclin mutants and by theknown or suspected substrates of cyclin–CDK complexes.

Overview of budding yeast cyclins and CDKsAs their name implies, cyclins were discovered as

proteins that oscillated in abundance through the cellcycle5. Following this discovery, experiments in variouseukaryotic cells firmly established cyclins as key regu-lators of the cell cycle that function by activating akinase partner, the CDK. As more cyclins were clonedand studied, it became apparent that not all cyclins showcell-cycle-dependent degradation and synthesis. How-ever, all cyclins share limited homology at the primary-sequence level in a region called the ‘cyclin box’6. Thecyclin-box homology reflects a structural motif, the‘cyclin fold’, required for interaction with and activationof the CDK partner2. Currently, admission to the cyclinfamily of proteins requires cyclin-box homology anddemonstrable interaction with a CDK.

There are 22 cyclins in budding yeast that bind to oneof five CDK enzymes: Cdc28p, Pho85p, Kin28p, Srb10pand Ctk1p (Table 1). Using multiple sequence alignment,a phylogenetic tree of budding yeast cyclins can becompiled. In general, the yeast cyclins fall into familiesthat reflect their CDK specificity as well as ‘subfamilies’that are more indicative of their function and regulation(Table 1, Fig. 1). Nine cyclins are known to activateCdc28p, the only CDK enzyme in budding yeast with anessential role in cell cycle progression. Other yeast CDKscontribute to cell-cycle progression through the regulationof gene expression and cell metabolism. Like Cdc28p, oneof these CDKs, Pho85p, associates with multiple cyclins7.In contrast, the three remaining yeast CDKs, Kin28p,

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66Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00PII: S0168-9525(97)01322-X

The cyclin family ofbudding yeast: abundantuse of a good ideaBRENDA ANDREWS (brenda.andrews@utoronto.ca)

VIVIEN MEASDAY (vivien.measday@utoronto.ca)

Cyclins are highly conserved proteins that activate cyclin-dependent kinases (CDKs) to regulate the cell cycle,transcription and other cellular processes. The completionof the genome sequence of the budding yeastSaccharomyces cerevisiae allows an appraisal of thefunctions of the entire complement of cyclins in aeukaryotic organism. The cyclin family of budding yeast isreviewed from a functional perspective with an emphasison what genetic and biochemical experiments haverevealed about cyclin–CDK substrates.

TABLE 1. S. cerevisiae cyclins and their CDKsa

CDK Cyclin Cyclin–CDK functionb

Cdc28p Cln1p, Cln2p, Cln3p Essential for START: spindle-pole-body duplication, budding and polarized growth, Sic1p proteolysis, inhibition of CLB proteolysis, activation of G1 transcription, acquisition of pheromone resistance

Clb5p, Clb6p DNA replicationClb1p, Clb2p, Clb3p, Clb4p Essential for mitosis: spindle assembly, inactivation of CLN transcription,

suppression of polarized bud growth, repression of SBF transcription factorPho85p Pcl1p Required for START in the absence of Cln1p, Cln2p

Clg1p, Pcl5p, Pcl9p UnknownPho80p Negative regulation of Pho4p transcription factor in response to phosphatePcl6p, Pcl7p UnknownPcl8p, Pcl10p Glycogen synthase kinase

Kin28p Ccl1p Transcription, CTD kinaseSrb10pc Srb11pd Transcription, CTD kinaseCtk1p Ctk2p Transcription, CTD kinase

aCyclin-dependent kinases.bFor reviews on budding yeast cyclin function, see Refs 4, 8, 9, 32. Pcl8p/10p–Pho85p has recently been identified as a glyco-gen synthase kinase (D. Huang et al., unpublished).cSrb10p is also known as Ume5p, Ssn3p and Are1p. dSrb11p is also known as Ssn8p and Ume3p (Ref. 8).

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Srb10p and Ctk1p, each associate with a single, dedicatedcyclin to regulate gene expression through close ties withthe transcriptional machinery8. Below, we survey the vari-ous branches of the loosely knit family of budding yeastcyclins from a functional perspective and with an empha-sis on known or genetically implicated substrates.

The Cdc28p cyclin familyCLNs: G1 cyclins

After a yeast cell is born, the first major task ofCdc28p-kinase complexes is to promote cell-cycle com-mitment once the cell reaches a critical size during lateG1 phase; this commitment phase of the cell cycle hasbeen termed ‘START’. The set of events that defineSTART include spindle-pole-body duplication, budding,initiation of DNA synthesis and repression of the signal-ling pathway that promotes mating4. Activation of Cdc28pat START requires the G1 cyclins Cln1p, Cln2p andCln3p. Two key genetic observations have establishedoverlapping roles for CLNs as essential dose-dependentregulators of START. First, the three CLN genes aregenetically redundant; strains deleted for any two CLNgenes have an altered cell size but divide more or lessnormally, while a strain lacking all three CLN genes arrestsbefore START. Second, CLN overexpression or mutationalactivation of a CLN accelerates START (see Table 2 andRefs 4, 9 for reviews of cln mutant phenotypes). How-ever, Cln3p is only distantly related to Cln1p and Cln2p,which show extensive similarity (Fig. 1). Indeed, Cln3pdiffers markedly from Cln1p and Cln2p in its transcrip-tional regulation and biochemical activity. CLN1/2 expres-sion (and Cln1/2p-associated kinase activity) is stronglycell-cycle regulated, with peak expression in late G1(Ref. 10). Periodic expression of CLN1/2 depends on theheterodimeric transcription factor SBF which, togetherwith a distinct factor called MBF, controls a program ofSTART-dependent gene activation11. Targets of SBF andMBF include other cyclins (see below), cell-wall biosyn-thetic genes12 and genes required for DNA synthesis11.In contrast, the CLN3 transcript and protein are presentthroughout the cell cycle, with increased CLN3 expressionoccurring early in G1 phase (Table 2)10,13.

Cln3p is also biochemically distinct from Cln1p andCln2p. Cln1p and Cln2p are more abundant than Cln3pand the kinase activity associated with Cln3p is lessactive in vitro10,14. There is also evidence that Cln3p hasa substrate specificity that is distinct from that of Cln1pand Cln2p in vivo (Table 2)10,15. Indeed, a large numberof genetic studies suggest that the Cln3p–Cdc28p kinaseis specialized to activate transcription through SBF andMBF, once cells reach a critical size. As a consequence,SBF/MBF target genes, including CLN1 and CLN2, areactivated. In contrast, Cln1p and Cln2p are dispensablefor gene activation but necessary for the timely execu-tion of other START-related events, such as budding,spindle-pole-body duplication and DNA synthesis(Table 2)10,14,16–18. How SBF and MBF are activated bythe Cln3p–Cdc28p kinase is not known. Components ofSBF and MBF might be substrates of Cln3–Cdc28p but,so far, mutations of cell-cycle-regulated phosphorylationsites on the two known components of SBF have failedto show regulation of SBF activity by Cdc28p (Refs 19,20). Alternatively, SBF and MBF might serve to localizeCln3p–Cdc28p kinase complexes to the promoters of

cell-cycle-specific genes. In this model, the importantsubstrates of Cln3p–Cdc28p might not be SBF/MBF but,rather, components of the transcriptional machinery.

Other aspects of START are reflected in the substratesof CLN–Cdc28p kinases. CLN kinases regulate the onsetof DNA replication by triggering destruction of Sic1p,an inhibitor of the CLB–Cdc28p-kinase complexes thatpromote DNA replication (see below and Fig. 2)21,22. Inthe absence of SIC1, CLN genes are no longer requiredfor viability22,23, suggesting that the only non-redundantrole for CLNs is to regulate Sic1p negatively. Indeed, thephosphorylation and destruction of Sic1p in vivo requiresCLN–Cdc28p (Refs 21, 22) as well as the Cdc34p, Cdc4p,Cdc53p and Skp1p proteins21,22,24. Cdc34p is a ubiquitin-conjugating enzyme, and a complex composed ofCdc34p, Cdc4p, Cdc53p and Skp1p is required for ubiqui-tination of phosphorylated Sic1p in vitro25,26. The modelis that Sic1p is phosphorylated by CLN kinases and,thereby, targeted for ubiquitin-mediated proteolysis,permitting the activation of CLB kinase and DNA repli-cation (see below and Refs 25, 26). CLN kinases also pro-mote the development of CLB kinase activity, throughthe repression of CLB proteolysis as cells pass throughSTART27. Whether components of the proteolytic machin-ery that degrades CLBs are substrates for CLN kinasesremains to be determined.

The regulation of Sic1p stability shows remarkableparallels to the regulation of another CDK inhibitor, Far1p,and of the CLN proteins themselves. Ubiquitination anddegradation of Cln2p in vivo, like that of Sic1p, requires

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396.9

350 300 250 200 150 100 50 0

Srb11pCcl1pCln3pCln1pCln2pClb4pClb3pClb6pClb5pClb1pClb2pCtk2pPho80pPcl10pPcl8pPcl7pPcl6pClg1pPcl5pPcl1pPcl9pPcl2p

FIGURE 1. Phylogenetic tree of budding yeast cyclins, based onmultiple sequence alignment of the most conserved region (the‘Pcl Box’; residues 45–110 of the cyclin box, see Refs 6, 7),generated using the Megalign program (DNASTAR). Thealignment was generated by the Jotun Hein method, using aPAM250 weight table. The scale bar indicates the relative distance on the tree in arbitrary units. Families of related cyclinsare highlighted by shading: Cdc28p cyclins are in the two palestgreys; Pho85p cyclins are in the two darkest shades of grey; and other cyclins are in black.

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Cdc28p-dependent phosphorylation and componentsof the Cdc34p pathway28,29. Mutations that inactivateCdc28p-phosphorylation sites in Cln2p stabilize the pro-tein in vivo and shorten G1 phase28. Similarly, the degra-dation of Far1p requires the Cdc34p-ubiquitination sys-tem and probably depends on prior phosphorylation bya CLN–Cdc28p kinase30,31. Far1p is an inhibitor of CLN–Cdc28p kinases that is required to arrest the cell cycle inG1 phase in response to mating pheromones. Targetedproteolysis of Far1p is important to allow recovery fromthe cell-cycle arrest induced by pheromone31.

So far, analysis of CLN substrates suggests that theprimary role of CLN kinases is to create conditions thatare permissive for CLB kinase activation at the appro-priate time32. One theme that has emerged is that CLNstarget proteins for ubiquitin-dependent proteolysis. Thesubstrates that reflect the role of CLN–Cdc28p kinases inpromoting polarized cell growth, budding and spindle-pole-body duplication remain to be identified. It will beinteresting to see if the theme of targeted degradationpromoted by CLNs holds true for other substrates ofthese kinases.

CLBs: S-phase and mitotic cyclinsAs discussed above, CLN kinases set the stage for the

activation of CLB–Cdc28p complexes, which then pro-mote DNA replication and mitosis (reviewed in Ref. 32).Due to differences in transcriptional controls, the CLBcyclins appear at different times but their patterns ofexpression overlap. The first CLB cyclins expressed arethe S-phase cyclins, Clb5p and Clb6p – activation of

CLB5/6 transcription requires the MBF transcription factorand occurs in late G1 phase11. The four mitotic cyclins,Clb1p–4p, then appear: Clb3p and Clb4p during S phase,and Clb1p and Clb2p during G2. Clb5p and Clb6p arerequired for the timely initiation of DNA replicationbecause, in their absence, S phase is delayed33–35. DNAreplication still occurs in a clb5 clb6 mutant because theother CLB cyclins, Clb1p–4p, can promote S phase(Table 2). Conversely, the S-phase cyclin, Clb5p, can pro-mote mitosis in a strain lacking CLB3 and CLB4 (whichhas no obvious phenotype)35. This genetic redundancymakes it difficult to assign specific roles to individualCLBs; they might have overlapping functions in wild-type cells or they might assume additional roles only inthe absence of other CLBs.

Obvious candidates for the S-phase-specific targetsof CLB–Cdc28p complexes are essential proteins for theactivation and proper timing of DNA replication. Theassembly at origins of the so-called pre-replication com-plex (pre-RC) is a prerequisite for the initiation of DNAsynthesis upon activation of the S-phase CLB kinases(reviewed in Ref. 36). The pre-RC contains at least threeprotein complexes: (1) ORC, a six-subunit complex thatis bound to origins throughout the cell cycle; (2) Cdc6p,an unstable protein that probably binds to ORC in latemitosis and is required for pre-RC formation; and (3) theMCM proteins (Mcm2p–Mcm7p) that are important fororigin firing and are loaded onto chromatin in lateM phase36–38. Cdc6p has been detected in complexeswith Cdc28p, Clb5p and Clb2p in yeast extracts, and anN-terminal fragment of Cdc6p can be phosphorylated by

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TABLE 2. Cdc28p cyclins

Transcription factors

Expression controlling Substrates/associated Selected mutantCyclin during cell cycle expression proteinsa phenotypesc

CLN mutantsCln1p Peaks in late G1 SBF Far1p, Sic1p, p41, p94b CLN3-1 Small cell size, pheromone Cln2p Peaks in late G1 SBF Far1p, Sic1p, p41, p94 resistance, reduced G1Cln3p Peaks at M/G1 Mcm1p Far1p, p45, SBF? cln3D Large cell size,

lengthened G1cln1Dcln2Dcln3D Arrest in G1 as large

unbudded cells

CLB mutantsClb1p Peaks in late G2 Mcm1p/SFF?Clb2p Peaks in late G2 Mcm1p/SFF Nap1p, Swi4p,

Cdc6p? 6Swi5pd clb1Dclb2D Arrests in G2 with fully form formed mitotic spindle

Clb3p Peaks in S phase ?Clb4p Peaks in S phase ? clb1–4D Arrests in G2, fails to form

bipolar mitotic spindlesClb5p Peaks in late G1 MBF Cdc6p? ORC? MCMs?Clb6p Peaks in late G1 MBF Cdc6p? ORC? MCMs? clb5Dclb6D Delay in S phase

clb1–6D Arrests with multiple buds and fails to initiate DNA replication

aSubstrates followed by ‘?’ indicate genetic or functional evidence exists to suggest the protein is a substrate.bp41, p45, p94 are unidentified substrates co-immunoprecipitated with CLN–Cdc28 complexes (Refs 10, 15).cSee Refs 3, 4, 9, 32 for more extensive discussion of mutant phenotypes associated with CLN and CLB deletion.dSpecific CLB kinase(s) responsible for phosphorylation of Swi5p in vivo is unknown.

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CLB-kinase complexes in vitro39,40.How the interaction with CLB ki-nases affects the function of Cdc6pis not clear. Besides activating DNAreplication, CLB kinases are alsorequired to block assembly of thepre-RC after initiation, thus prevent-ing the inappropriate reinitiation of DNA replication41. Crosslinkingstudies have shown that the activityof CLB kinases prevents loading ofMcm7p onto origins outside of G1phase38. The ‘conflicting’ roles of S-phase CLB–Cdc28p complexesensure that DNA replication occursonly once per cell cycle. Preciselywhich components of the pre-RCare targeted by CLB kinases, to pro-mote DNA replication and to inhibitpre-RC complex formation, remainsto be determined.

Cells lacking CLB1, CLB2, CLB3and CLB4 initiate DNA replicationwith wild-type kinetics but fail toform bipolar mitotic spindles andarrest in G2 phase (Table 2; Refs 42,43). This arrest phenotype suggeststhat Clb1p, Clb2p, Clb3p and Clb4pare not normally required for DNAreplication, but that they are nec-essary for assembly or maintenanceof an intranuclear spindle. CLB activ-ity is also required to turn off the polarized bud growththat is initiated at START by the G1 cyclins44. The timingof their expression (Table 2) suggests that Clb3p andClb4p probably function in early spindle assembly dur-ing S phase, while Clb1p and Clb2p promote spindleelongation during mitosis. Indeed, clb1 clb2 doublemutants are inviable and arrest with a fully formedmitotic spindle, whereas a diploid strain lacking CLB1,CLB2 and CLB3 arrests with a short spindle42,45,46. Nap1pinteracts specifically with Clb2p and is a good candidatefor a Clb2p-kinase substrate involved in both spindlefunction and in repression of polarized cell growth47.Other substrates for CLB kinases, related to their rolesin spindle function and bud growth, are unknown butprobably include spindle-pole-body components, motorproteins needed for spindle elongation and regulatorsof spindle assembly and polarized cell growth18.

Two apparent targets of CLB kinases are cell-cycle-regulated transcription factors. One of these factors,Swi5p, accumulates in the cytoplasm before the end ofanaphase and becomes nuclear during G1 phase.Accumulation of Swi5p in the cytoplasm requiresCdc28p-dependent phosphorylation of its nuclear local-ization signal – inactivation of CLB kinases might allowentry of Swi5p into the nucleus, where it activates theexpression of a number of genes, including that encod-ing the CLB-kinase inhibitor, SIC1 (Refs 48–50). A secondtranscription factor that is probably regulated by CLBkinases is Swi4p, a component of the G1-specific tran-scription factor, SBF (see CLN section above). Repressionof SBF-regulated genes during G2 phase depends onCLB1, CLB2, CLB3 and CLB4, and association of Swi4p

with Clb2p can be detected in mitotically arrested cells43.It has been proposed that the association of Swi4p withmitotic forms of Cdc28p leads to inhibition of SBF activ-ity. Interestingly, the transcription of CLB1 and CLB2depends on CLB-kinase activity, suggesting that a thirdtranscription factor complex, Mcm1p/SFF, which acti-vates CLB2 gene expression, might be a substrate forCLB kinases in G2 phase51 (Fig. 2).

The Pho85p cyclin familyPHO85 encodes a non-essential CDK with 51% iden-

tity to Cdc28p and it has emerged as an importantmodel for the role of CDKs in processes beyond cell-cycle control. Pho85p was first discovered because of itsrole in regulating the activity of secreted acid phosphat-ases but has since been shown to play a broader role incell metabolism and division (reviewed in Ref. 52). Tengenes that encode known or putative Pho85p cyclinshave been identified7 and the pleiotropic nature ofPho85p function has been ascribed to its associationwith multiple cyclin partners.

Pho80p subfamilyThe Pho85p cyclins, or PCLs, have been grouped

into two subfamilies based on sequence similarity withinthe cyclin box region (Fig. 1, Table 3)7. Pho80p was thefirst protein to be identified as a Pho85p cyclin53 andconstitutes the founding member of the Pho80p ‘sub-family’. Pho80p–Pho85p complexes control expressionof the major acid phosphatase gene, PHO5, throughrepression of the transcription factor Pho4p (Ref. 53).When phosphate is abundant, Pho4p is phosphorylated

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Cell cycle Cell metabolism

Transcription

Ccl1p–Kin28p

Srb11p–Srb10p

Ctk2p–Ctk1p

RNAPol IICTD

SBF/MBF

Clb5p/6p–Cdc28p Cln1p/2p–Cdc28p(Pcl1p/2p–Pho85p)

Sic1p

Far1pCLNs

Budding

DNAreplication

ORCCdc6pMCMs

Clb1p–4p–Cdc28p

Mitotic events

Cln3p–Cdc28p

CLB–Cdc28p

Spindle-pole-bodyduplication

Swi4pSwi5p

SFF/Mcm1p

Pheromonepathway

CLBproteolysis

Pho80p–Pho85p

Pho4p

Pcl8p/10p–Pho85p

Gsy2p

FIGURE 2. Regulatory pathways involving yeast CDK complexes. Only those CDKcomplexes with established biological functions are shown. Cdc28p complexes are shownin yellow, Pho85p complexes in blue and other CDK complexes in purple. Knownsubstrates are in red text and proteins implicated as CDK targets in vivo are in green.Black arrows indicate activation of the target protein by the CDK and a black bar denotesrepression. See text and Tables 1–3 for discussion of these and other yeast CDKs.

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by Pho80p–Pho85p and is largely cytoplasmic, causingrepression of PHO5 expression54. In contrast, Pho4p isnuclear in pho80 or pho85 mutants, as is Pho4p, whichlacks Pho80p–Pho85p phosphorylation sites54. Theseobservations suggest that Pho80p–Pho85p complexesrepress PHO5 gene expression by reducing the concen-tration of nuclear Pho4p. However, strains expressing amutant of Pho4p that is constitutively nuclear are notfully constitutive for PHO5 expression54 – two-hybriddata suggest that Pho80p might also regulate Pho4p bybinding and masking its activation domain55. Whencells are starved of phosphate, Pho80p–Pho85p isrepressed by the CDK inhibitor Pho81p (Refs 56, 57).Pho81p contains six copies of a protein motif, theankyrin domain, which is found in mammalian CDKinhibitors; alone, the ankyrin repeats are sufficient toinhibit Pho80p–Pho85p kinase activity57,58.

The Pho80p–Pho85p-dependent phosphorylationsites on Pho4p have been mapped54 and reveal a con-sensus site for phosphorylation (Ser/Thr-Pro-x-Leu/Ile)that differs from the consensus phosphorylation site forCdc28p and other CDKs (Ser/Thr-Pro-x-Lys/Arg). Ingeneral, Pho85p kinases are likely to have a substratespecificity that is distinct from Cdc28p kinases – whileHistone H1 is a good substrate for Cdc28p kinases invitro, all forms of the Pho85p kinase examined so farfail to phosphorylate Histone H1 (Refs 53, 59, 60).

Recent studies have identified glycogen synthase(Gsy2p) as a second substrate for Pho85p in vivo61,62.Excess glycogen storage in pho85 mutants correlates withhyperactivation of glycogen synthase, which is knownto be inactivated by phosphorylation. Two relatedmembers of the Pho80p subfamily, Pcl8p and Pcl10p,appear to activate Pho85p specifically in its role as aglycogen-synthase kinase. Like pho85 mutants, a pcl8

pcl10 double mutant hyperaccumulates glycogen owingto increased activity of glycogen synthase, althoughexpression of the gene encoding acid phosphatase isunaffected (D. Huang, W. Wilson and P. Roach, unpub-lished). Furthermore, Pcl10p–Pho85p complexes phos-phorylate Gsy2p in vitro but do not phosphorylatePho4p, while Pho80p–Pho85p complexes have a lowactivity towards Gsy2p but efficiently phosphorylatePho4p (J. Moffat and B. Andrews, unpublished). Thus,Pcl10p and Pho80p provide an example of a yeast CDKacquiring a unique substrate preference, both in vitro andin vivo, when complexed with different cyclin subunits.

The remaining members of the Pho80p subfamily,Pcl6p and Pcl7p, are highly related cyclins whose func-tions are presently unknown.

Pcl1p/2p subfamilyThe two founding members of the Pcl1p/2p subfam-

ily appear to be involved in the regulation of the G1phase of the cell cycle. Although Pho85p is not essentialfor viability, it is required for G1 progression in the ab-sence of the Cdc28p cyclins CLN1 and CLN2 (Refs 59,60). Similarly, deletion of PCL1 and PCL2 causes G1arrest when CLN1 and CLN2 are also absent (Table 3)60.A role for Pcl1p/2p–Pho85p kinases in G1 phase is sug-gested by these genetic findings and supported by otherobservations. First, expression of PCL1 and PCL2, likethat of CLN1 and CLN2, is controlled by SBF and peaksat START. Second, Pcl2p–Pho85p kinase activity peaks inG1 phase and phosphorylates two co-immunoprecipitatedproteins, p75 and p85 (Table 3)60. Third, overexpressionof PCL1 or PCL2 (but not the CLN genes) rescues thelysis defect seen in strains mutated for components ofthe Slt2 MAP-kinase pathway63. Slt2p is required to main-tain cell integrity during periods of highly polarized cell

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TABLE 3. Pho85p cyclins

Transcription factors

Expression during controlling In vitro In vivo MutantCyclin cell cycle expression substratec substrated phenotypese

Pcl1p Peaks in late G1 SBF Pho4p Pcl1p pcl1Dpcl2D Synthetically lethalwith cln1Dcln2D

Pcl2p Peaks in late G1 SBF, Swi5pb Pho4p p75, p85 pcl1Dcln1Dcln2D Inviable as a diploidand arrests in G1

Pho80p Constant Pho4p Pho4p, Pho80p pcl1,2,5,9,clg1D Elongated buds andconnected chains ofcells in a diploid

Clg1p ConstantPcl5p Increase at G1/S SBF?a pho80D Constitutive PHO5

expression, slowgrowth on glycerol,acetate and ethanol

Pcl6p Constant Pho4pPcl7p Constant Pho4pPcl8p Constant Gys2p? Gys2pPcl9p Peaks in late M/earlyG1 Swi5pb Pho4p pcl8Dpcl10D Hyperaccumulate

glycogenPcl10p Constant Gys2p Gys2p

aThe PCL5 promoter has SCB elements but it is not known whether PCL5 expression is dependent on SBF (Ref. 7).bPCL9 expression is dependent on the Swi5p transcription factor70,71 as is PCL2 expression70.c–eSee text for discussion and references.

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growth. Polarized cell growth is important for budemergence and response to pheromone, both of whichare early cell-cycle events (reviewed in Ref. 18). Thecellular targets of Pcl1p/2p–Pho85p kinases are unknown,but they are probably different from CLN-kinase sub-strates because the substrate specificities of Pho85p andCdc28p appear to be distinct.

Three other members of the Pcl1p/2p subfamily(Clg1p, Pcl5p and Pcl9p) have been identified (Table 3).Studies of PCL9 expression provide further support fora role for Pho85p kinases in cell-cycle progression. Ex-pression of PCL9 is dependent on Swi5p and, like otherSwi5p-regulated genes, peaks in late mitosis/early G1phase (Table 3; Refs 70, 71). Genetic studies of strainsdeleted for members of the Pcl1p/2p subfamily haverevealed little about the function of these cyclins.However, a diploid strain lacking the entire Pcl1p/2psubfamily shows pronounced morphological defectsincluding elongated buds and connected chains of cells7.Perhaps Pcl1p/2p–Pho85 kinases normally participate inprocesses related to polarized cell growth and budemergence, a contribution that becomes essential whenG1 progression is compromised.

Pho85p is most similar to mammalian CDK5 which,like Pho85p, appears to have roles beyond cell-cycle con-trol. CDK5 is complexed with an activating subunit, p35,in postmitotic neuronal cells64. Interestingly, p35–CDK5might regulate cell morphogenesis and cytoskeletaldynamics through phosphorylation of neurofilaments inpostmitotic neurons64. p35 does not show sequencehomology with PCLs or other cyclins and could repre-sent a non-cyclin-activating subunit for CDK5. However,Pcl homologues have been found in Caenorhabditiselegans (CE00385; GenBank L15313), Neurospora crassa(Q06712; SWISS-PROT) and Schizosaccharomyces pombe(C19E9; pombe database) suggesting that PCL functionmight be conserved in other organisms.

Distant relatives: other yeast cyclins and transcriptionThree cyclin–CDK complexes in budding yeast have

been implicated as kinases that phosphorylate the C-terminal domain (CTD) of RNA polymerase II (pol II)in vivo (reviewed in Ref. 8). Two of these cyclin–CDKpairs, Ccl1p–Kin28p and Srb11p–Srb10p, are associatedwith the yeast polII holoenzyme; Ccl1p–Kin28p is thecyclin–CDK component of the general transcription factorTFIIH, and Srb11p–Srb10p is found in the holoenzymecomplex65,66. Mutations in the KIN28 and SRB10 geneseach reduce phosphorylation of the CTD in vivo66,67 andare associated with a variety of defects in gene expres-sion68. A more divergent cyclin–CDK complex has beenisolated that phosphorylates the RNA pol II CTD but isnot detectable in purified yeast holoenzyme. This com-plex is composed of the cyclin-C-like protein Ctk2p, aCDK-related protein Ctk1p and a protein with no signifi-cant similiarity to other proteins, Ctk3p (Ref. 69). Theidentification of CDKs as components of the RNA pol IIholoenzyme and as CTD kinases reflects a more generalrole for CDKs in controlling transcription. The binding ofcyclin to components of the basal-transcription machinerymight provide a means of coordinating mRNA synthesiswith cell division and other cellular processes. In othercases, CDKs might be recruited to promoters by activatorproteins, thereby stimulating transcription (see discussion

of SBF above). Both of these models invoke a broaderrole for CDKs in the regulation of gene expression.

Concluding remarksRemarkable progress has been made in the last few

years towards an understanding of how cyclins regulateCDK activity in the control of cell-cycle progression.Studies in yeast have been particularly important inhighlighting a role for CDKs, not only in cell cycle con-trol but also in transcription and cellular metabolism(summarized in Fig. 2). Also, the yeast-genome project hastreated us to the first view of a complete set of eukary-otic cyclins. The broad application of the cyclin–CDKparadigm might surprise even the most ardent CDKenthusiast. If each cyclin is strictly faithful to a given CDK,there are 22 cyclin–CDK complexes in yeast. However,mammalian cyclins are known to activate more than oneCDK subunit and the same might be true of yeast CDKs.

Have we identified all yeast cyclins? The crystalstructure of cyclin A revealed a second ‘cyclin fold’, C-terminal to that derived from the conserved cyclin boxmotif, yet this second domain is only 13% identical tothe cyclin box (reviewed in Ref. 2). Perhaps once weknow how to look, we will recognize more yeast pro-teins as members of the cyclin family.

What is the basis of cyclin–CDK specificity? The ideathat cyclins can target CDK activity by binding specificsubstrates is supported by studies of mammalian andyeast CDKs. Little is known about the subcellular localiz-ation of yeast cyclins and, as highlighted in our review,relatively few proteins have been established as bonafide CDK substrates. Biochemical efforts and sophisticatedgenetic experiments should allow identification of morecyclin–CDK substrates and reconstitution of cyclin–CDKcomplexes in vitro. With these tools in hand, the bindingof different cyclins to CDKs and the interactions of cyclinsand substrates can be assessed. In this way, we will con-tinue to expand our view of how cyclin–CDK complexesregulate cell division, transcription and metabolism.

AcknowledgementsWe thank S. Climie, C. Goding, L. Johnston, P. Roach,

A. Spence and M. Tyers for comments on the manuscript.We are grateful to L. Moore for generating the cyclinalignment and to R. Zamel for help with the figures. Wethank R. Deschaies, C. Goding, W. Harper, L. Johnston,M. Mendenhall, M. Peter, P. Roach and M. Tyers for com-municating their unpublished results. B.J.A. is supportedby the Medical Research Council (MRC) of Canada, fundsfrom the MRC and Apotex Inc. through the University/Industry Program and the National Cancer Institute ofCanada. V.M. was partially supported by an OntarioGraduate Scholarship.

References1 Nigg, E.A. (1995) BioEssays 17, 471–4802 Morgan, D.O. (1996) Curr. Opin. Cell Biol. 8, 767–7723 Deschaies, R.J. (1997) Curr. Opin. Genet. Dev. 7, 7–164 Nasmyth, K. (1993) Curr. Opin. Cell Biol. 5, 166–1795 Evans, T. et al. (1983) Cell 33, 389–3966 Hunt, T. (1991) Semin. Cell Biol. 2, 213–2227 Measday, V. et al. (1997) Mol. Cell. Biol. 17, 1212–12238 Poon, R.Y.C. and Hunter, T. (1995) Curr. Biol. 5,

1243–12479 Futcher, B. (1996) Yeast 12, 1635–1646

TIG FEBRUARY 1998 VOL. 14 NO. 2

71

REVIEWS

10 Tyers, M., Tokiwa, G. and Futcher, B. (1993) EMBO J. 12,1955–1968

11 Breeden, L. (1995) Curr. Top. Microbiol. Immunol. 208,95–127

12 Igual, J.C., Johnson, A.L. and Johnston, L.H. (1996) EMBOJ. 15, 5001–5013

13 McInerny, C.J. et al. (1997) Genes Dev. 11, 1277–128814 Levine, K., Huang, K. and Cross, F.R. (1996) Mol. Cell.

Biol. 16, 6794–680315 Tyers, M. and Futcher, B. (1993) Mol. Cell. Biol. 13,

5659–566916 Dirick, L., Bohm, T. and Nasmyth, K. (1995) EMBO J. 14,

4803–481317 Stuart, D. and Wittenberg, C. (1995) Genes Dev. 9,

2780–279418 Lew, D.J. and Reed, S.I. (1995) Curr. Opin. Genet. Dev. 5,

17–2319 Koch, C. and Nasmyth, K. (1994) Curr. Opin. Cell Biol. 6,

451–45920 Sidorova, J.M., Mikesell, G.E. and Breeden, L. (1995) Mol.

Biol. Cell 6, 1641–165821 Schwob, E., Bohm, T., Mendenhall, M.D. and Nasmyth, K.

(1994) Cell 79, 233–24422 Schneider, B.L., Yang, Q.H. and Futcher, B. (1996) Science

272, 560–56223 Tyers, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,

7772–777624 Bai, C. et al. (1996) Cell 86, 263–27425 Feldman, R.M.R., Correll, C.C., Kaplan, K.B. and

Deschaies, R.J. (1997) Cell 91, 221–23026 Skowyra, D. et al. (1997) Cell 91, 209–21927 Amon, A., Irniger, S. and Nasmyth, K. (1994) Cell 77,

1037–105028 Lanker, S., Valdivieso, M.H. and Wittenberg, C. (1996)

Science 271, 1597–160129 Willems, A. et al. (1996) Cell 86, 453–46330 McKinney, J.D., Chang, F., Heintz, N. and Cross, F.R.

(1993) Genes Dev. 7, 833–84331 Henchoz, S. et al. (1997) Genes Dev. 11, 3046–306032 Nasmyth, K. (1996) Trends Genet. 12, 405–41233 Epstein, C.B. and Cross, F.R. (1992) Genes Dev. 6,

1695–170634 Kuhne, C. and Linder, P. (1993) EMBO J. 12, 3437–344735 Schwob, E. and Nasmyth, K. (1993) Genes Dev. 7,

1160–117536 Stillman, B. (1996) Science 274, 1659–166437 Jallepalli, P.V. and Kelly, T.J. (1997) Curr. Opin. Cell Biol.

9, 358–36338 Tanaka, T., Knapp, D. and Nasmyth, K. (1997) Cell 90,

649–66039 Piatti, S. et al. (1996) Genes Dev. 10, 1516–153140 Elsasser, S. et al. (1996) Mol. Biol. Cell 7, 1723–173541 Dahmann, C., Diffley, J.F.X. and Nasmyth, K.A. (1995)

Curr. Biol. 5, 1257–126942 Fitch, I. et al. (1992) Mol. Biol. Cell 3, 805–81843 Amon, A., Tyers, M., Futcher, B. and Nasmyth, K. (1993)

Cell 74, 993–100744 Lew, D.J. and Reed, S.I. (1993) J. Cell Biol. 120, 1305–132045 Surana, U. et al. (1991) Cell 65, 145–16146 Richardson, H. et al. (1992) Genes Dev. 6, 2021–203447 Kellogg, D. and Murray, A.W. (1995) J. Cell Biol. 130,

675–68548 Moll, T. et al. (1991) Cell 66, 743–75849 Knapp, D., Bhoite, L., Stillman, D.J. and Nasmyth, K.

(1996) Mol. Cell. Biol. 16, 5701–570750 Toyn, J.H. et al. (1996) Genetics 145, 85–9651 Maher, M. et al. (1995) Mol. Cell. Biol. 15, 3129–313752 Lenburg, M.E. and O’Shea, E.K. (1996) Trends Biochem.

Sci. 21, 383–38753 Kaffman, A., Herskowitz, I., Tjian, R. and O’Shea, E.

TIG FEBRUARY 1998 VOL. 14 NO. 2

72

(1994) Science 263, 1153–115854 O’Neill, E.M., Kaffman, A., Jolly, E.R. and O’Shea, E.K.

(1996) Science 271, 209–21255 Jayaraman, P.S., Hirst, K. and Goding, C.R. (1994) EMBO J.

13, 2192–219956 Hirst, K., Fisher, F., McAndrews, P.C. and Goding, C.R.

(1994) EMBO J. 13, 5410–542057 Schneider, K.R., Smith, R.L. and O’Shea, E.K. (1994)

Science 266, 122–12658 Ogawa, N. et al. (1993) Mol. Gen. Genet. 238, 444–45459 Espinoza, F.H., Ogas, J., Herskowitz, I. and Morgan, D.O.

(1994) Science 266, 1388–139160 Measday, V. et al. (1994) Science 266, 1391–139561 Timblin, B.K., Tatchell, K. and Bergman, L. (1996)

Genetics 143, 57–6662 Huang, D., Farkas, I. and Roach, P.J. (1996) Mol. Cell.

Biol. 16, 4357–436563 Madden, K. et al. (1997) Science 275, 1781–178464 Lew, J. and Wang, J.H. (1995) Trends Biochem. Sci. 20,

33–3865 Feaver, W.J., Svejstrup, J.Q., Henry, N.L. and Kornberg,

R.D. (1994) Cell 79, 1103–110966 Liao, S.M. et al. (1995) Nature 374, 193–19667 Valay, J.G. et al. (1995) J. Mol. Biol. 249, 535–54468 Cismowski, M.J., Laff, G.M., Solomon, M.J. and Reed, S.I.

(1995) Mol. Cell. Biol. 15, 2983–299269 Sterner, D.E., Lee, J.M., Hardin, S.E. and Greenleaf, A.L.

(1995) Mol. Cell. Biol. 15, 5716–5724

References added in proof70 Aerne, B.L., Johnson, A.L., Toyn, J. and Johston, L. Mol.

Biol. Cell (in press)71 Tennyson, C.N., Lee, J. and Andrews, B. Mol. Microbiol.

(in press)

B. Andrews and V. Measday are in the Department ofMolecular and Medical Genetics, University of Toronto, 1 Kings College Circle, Toronto, Canada M5S 1A8.

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