INTRODUCTION

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Saccharomyces cerevisiae possesses five cyclin-dependent protein kinases (CDKs) (Cdc28, Pho85, Kin28, Ssn3, and Ctk1), but Cdc28, the subject of this review, is the best studied by far. Cdc28 is the central coordinator of the major events of the yeast cell division cycle. Environmental effects that influence the decision to undergo cell division or the fidelity and rate of key mitotic events ultimately affect Cdc28 kinase activity. This review strives to provide a comprehensive survey of the published literature on how Cdc28 activity is generated and regulated. There have been many excellent shorter reviews of various aspects of this system in the last few years, and they provide an ideal general introduction to various aspects of the yeast cell cycle and opportunities for looking at specific topics in depth. The long-review format of Microbiological and Molecular Biology Reviews allows us to present a more exhaustive summary that we hope will be of use to our coworkers and will serve as a secondary source for those already familiar with basic yeast physiology. Discussion of the functions of the CDKs is kept to a minimum, except for the (numerous) instances when CDKs act as CDK regulators. Likewise, a discussion of the many homologous genes and gene products from other species is minimized or omitted; it is used mostly to help make sense of regulatory modes that are well worked out in other systems but not in S. cerevisiae. This compromise was necessary to limit what is already a voluminous topic, and we apologize to the many investigators whose work anticipated and inspired the parallel work in budding yeast but that we were unable to cite.

Instead of conducting a gene-by-gene summary or a walk through the cell cycle, we have chosen to organize the topics in this review by starting with a short description of key Cdc28 regulators (cyclins, CDK inhibitors [CKIs], the enigmatic Cks1, and phosphorylation of Cdc28) and then organizing the influences on these regulators by large-scale process starting with transcription and ending with proteolysis. Finally, the effects of environmental influences on these processes and regulators are discussed.

Many of the genes discussed have been identified by multiple laboratories over a long span of time and have consequently acquired multiple labels. To simplify the discussion, we use the gene names favored by the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces) and Proteome (www.proteome.com). Aliases for these genes can be found at the Saccharomyces Genome Database and Proteome Web sites and in Table 1. Table 1 also contains a short synopsis of the function of each gene and the positions of important domains discussed in the text. Standard S. cerevisiae genetic conventions are used throughout (dominant or wild-type genes and their mRNAs are in capital italics, recessive mutants are in lowercase italics, and  refers to a gene deletion or disruption; e.g. CLN3 is wild type, CLN3-1 is a dominant mutant allele, cln3 is a recessive mutant allele, and cln3 is a deletion). Superscripts are added to denote alleles with specific properties: cln3^ts is a temperature-sensitive allele, and CLN3^stab encodes a protein that is hyperstable relative to the wild-type protein. The protein products of a particular gene are in roman type (Cln3 is the gene product of CLN3 and Cln3-1 is the product of CLN3-1 allele). Genes under the transcriptional control of heterologous promoters are designated, e.g., GAL1pCLN3, which indicates that the promoter element from the GAL1 gene is used to control expression of the open reading frame (ORF) for CLN3. Protein fusions are indicated with superscripts indicating the region of the protein that is present: Cln3^404-488--Gal is a fusion of Cln3 residues 404 to 488 to -galactosidase.

General CDK Principles and IssuesCDKs. As the name implies, the CDKs are protein kinases that are dependent for their activity on the binding of a cyclin subunit (for general reviews, see references 390 and 440). A large amount of useful information about CDKs and protein kinases in general can be obtained at www.sdsc.edu/Kinases/. The CDK catalytic subunits are generally recognized by a shared high degree of sequence identity with other members of the family (218), particularly in a domain near the N terminus known as the PSTAIRE motif. CDKs were first discovered during the genetic analyses of the cell cycles of budding (227, 351, 403) and fission (240, 410) yeasts, and, in landmark studies, a CDK was found to be a component of Xenopus mitosis promoting factor (MPF; known as maturation-promoting factor at that time) (144, 348). Eukaryotic cells generally possess multiple CDKs that are involved in a wide range of activities. For historical reasons, the CDK most involved in M phase initiation is called Cdc2 in most organisms (325), but is Cdc28 in S. cerevisiae. In the fungi, the other CDKs have names based on their phenotypes, but in most other systems the CDKs are labeled Cdk2, Cdk3, etc., based on their order of discovery in mouse or human systems. Cdk1 is seeing increased usageit is equivalent to Cdc2.

Activation by cyclins. Cyclins were discovered biochemically as proteins that appeared and disappeared in synchrony with early embryonic cleavage divisions in sea urchins (168) and genetically in yeast for their cell cycle effects (45, 82, 214, 402, 537). The realization in 1989 that cyclins were complexed with CDKs in diverse eukaryotes (46, 140, 193, 379, 617) marked the birth of the modern era in cell cycle research. Most organisms possess multiple cyclins and CDKs, and although cyclin-CDK interactions are specific, CDKs can be activated by multiple cyclins and cyclins can activate multiple CDKs (21, 496). (As yet, there is no example in S. cerevisiae of a cyclin activating more than one kinase, however [14].)

Activation by phosphorylation. Full activation of most CDK-cyclin complexes requires phosphorylation in the T loop at the position corresponding to T169 of Cdc28 (120, 142, 207, 521). In the crystal structure of the phosphorylated Cdk2-cyclin A complex, phosphorylation of Cdk2 T160 (equivalent to Cdc28 T169) results in additional movement of the T-loop, opening up the protein substrate binding region and increasing the number of contacts between the Cdk and the cyclin (472). The T-loop is a site for autophosphorylation in many protein kinases but not in CDKs. Phosphorylation at this position in a CDK requires a CDK-activating kinase (CAK). The first CAKs to be identified were purified from animal cells and are themselves CDKs, consisting of Cdk7 (177, 444, 520), cyclin H (180, 365) and, in some circumstances, a third protein, Mat1 (124, 179, 550). The Cdk7-cyclin H-Mat1 complex is also a component of the general transcription factor TFIIH (468, 489, 498), which phosphorylates the long carboxy-terminal domain (CTD) of RNA polymerase II and participates in transcription initiation and nucleotide excision repair. This pattern is not invariant, however, as results of the studies of CAK activity in S. cerevisiae made clear. The S. cerevisiae homologs of Cdk7, cyclin H, and Mat1 are Kin28, Ccl1 (586), and Rig2 (171), respectively, but although Kin28-Ccl1-Rig2 is a component of TFIIH and phosphorylates the CTD repeat of RNA polymerase II (172, 545, 587), Kin28-Ccl1-Rig2 does not possess CAK activity (93). As discussed below, the true CAK in S. cerevisiae is not a CDK and is not a component of TFIIH (see "Stimulatory phosphorylation on T169") (165, 275, 560). Based on a very small sample, it appears that plants resemble the budding-yeast pattern (585) while Schizosaccharomyces pombe CAK has animal-like features (66, 116). These differing patterns seem to reveal an early evolutionary split in the manner in which eukaryotes handle CTD versus CDK phosphorylation events.

Inhibition by CKIs. Opposing the action of the cyclins are the CKIs. These were first described genetically (83) and biochemically (380) in S. cerevisiae. Recognizable homologs of the yeast Cdc28 inhibitors have yet to be identified in metazoans. The mammalian CKIs (for a review, see reference 497) have received extensive attention due to their roles as tumor suppressors and developmental regulators. These CKIs are grouped into two major classes based on shared structural features and biochemical function. Members of the INK4 class are characterized by the presence of multiple 32- to 33-residue "ankyrin repeats" (49). These CKIs bind to and inhibit a small subset of CDKs (Cdk4 and Cdk6) (490), free or in complex with a cyclin, that are primarily responsible for promoting passage through G₁. Crystal (594) and nuclear magnetic resonance spectroscopy (356) structures of two members of this class have been published. The budding-yeast Pho81, inhibitor of the Pho80-Pho85 cyclin-CDK complex, is structurally similar to the INK4 proteins (243, 416, 481). Members of the second class of mammalian CKIs are general CDK inhibitors and recognize both CDK and cyclin components. Analysis of this class is complicated by the observations that (i) the founding member, p21^Cip1/Waf1, is a CDK-cyclin assembly factor at low concentrationsand thus a CDK activatorand inhibits only at higher concentrations (635) and (ii) that p21^Cip1/Waf1 also binds and inhibits proliferating-cell nuclear antigen (637). No member of this class has been identified in budding yeast.

Inhibition by phosphorylation. Phosphorylation on the CDK catalytic subunit at positions corresponding to T18 (306, 408) and Y19 (208) of Cdc28 inhibits the activity of CDKs (for a review, see reference 38). In vertebrate systems, both phosphorylations are needed for maximal CDK inhibition (306, 408). In fungal systems, tyrosine phosphorylation alone seems sufficient to meet known regulatory needs, although phosphorylation at the position corresponding to T18 has been detected (9, 119). The side chains of T18 and Y19 are near the ATP binding site, but the mechanism by which phosphorylation at these sites inhibits the CDK activity has not been established. Phosphorylation of Y15 in human Cdc2 (equivalent to Cdc28 Y19) does not significantly alter the K_m for ATP (25). It is postulated that the positioning of the ATP  phosphate or of active-site residues may be disrupted by Y19 phosphorylation, but it is also possible that interactions between the CDK and its protein substrates or other interacting factors are affected. Inhibitory phosphorylation on Cdc2 is associated with checkpoints preventing entry into M phase due to incomplete DNA synthesis (159), to unrepaired DNA damage (267, 286, 626), or to intrinsic cell cycle requirements such as cell size (147, 208). Tyrosine phosphorylation on other CDKs is also important for regulating other cell cycle transitions (258, 473, 557) but has been studied to a lesser extent. These phosphorylation events are controlled by multiple, dually specific protein kinases and phosphatases which, in turn, are under complex and not well-understood controls (for reviews, see references 38, 266, 330, and 568).

The first mutant allele of CDC28 was originally isolated in the early 1970s (226, 228, 229) and was quickly recognized as an important integrator of external controls on cell cycle events (225). The recognition that it encoded a protein kinase (351, 457) whose activity was cell cycle regulated (382, 615), that was activated by cyclins (214, 462, 614), and that was highly conserved in eukaryotic evolution (35, 240, 325) came over a 10-year span in the 1980s. Cdc28 is now recognized as the central component of an elaborate mechanism that controls the timing of events in the yeast cell cycle.

The gene encoding Cdc28 is essential. Most of the original cdc28 mutants arrest cell cycle progression at Start when shifted to restrictive conditions (225, 383, 455, 456), but alleles with other phenotypes are known. A few alleles, cdc28-1N being the most prominent, arrest predominantly in G₂ (439, 541). This late cell cycle arrest can also be observed with many of the Start-arrest alleles when the restrictive conditions are applied to cells shortly after Start (458). Defects in postmating nuclear fusion (146), mitotic chromosome stability (125), mitochondrial DNA transmission (125), radiation sensitivity (304), spindle pole body separation (341), and meiosis (507) have also been attributed to defects in Cdc28 function.

Although its protein kinase activity is under multiple, complex controls, the abundance of the Cdc28 polypeptide is virtually unchanged throughout the cell cycle (382). Very little has been reported on environmental or cell cycle effects on Cdc28 transcription, but the protein product is stable (40) and naturally occurs in excess (75, 617). Constitutive overproduction of wild-type Cdc28 also seems to be tolerated relatively well by the cell (383, 454), and so transcriptional and translational regulation of Cdc28 has not been considered important. Virtually all of the controls on Cdc28 activity are manifested at the posttranslational level and are detailed in the remainder of this review.

It is often but erroneously stated that S. cerevisiae has a single CDK. It is appropriate at this point to emphasize that in addition to Cdc28, four other budding-yeast CDKs are known (Table 2). Three of theseKin28 (93, 587), Ssn3 (309, 339), and Ctk1 (324), in association with their cyclin activators Ccl1 (545, 586), Ssn8 (309, 339), and Ctk2 (530), respectivelyphosphorylate the carboxy-terminal repeat domain of RNA polymerase II and thus play a role in transcription. The remaining CDK, Pho85, is activated by a complex family of at least 10 cyclins (see reference 14 for a review). The functions of this CDK are still being delineated, but they include roles in the regulation of phosphate and glycogen metabolism. Of special interest for this review, the Pho85-Pcl1 and Pho85-Pcl2 complexes play a role in G₁ passage. Both cyclin components are periodically expressed late in G₁ (414, 578), and deletions of the genes encoding these cyclins or of PHO85 are synthetically lethal with deletions of CLN1 and CLN2 (166, 377), late G₁ activators of CDC28 (see "G₁ cyclins"). Another cyclin activator of Pho85, Pcl9 is periodically expressed at the M/G₁ border and may play a role in cell cycle regulation as well (556). Pcl1, Pcl2, and Pcl9 belong to a subfamily of Pho85 cyclins (the other members are Clg1 and Pcl5) that play a role in the determination of bud site selection (556). As yet, there is no indication that any of these complexes regulate Cdc28 activity, and they are not discussed further in this review.

Historically, Cdc28 cyclins have been classified into two broad groups: the three G₁ cyclins (Cln1 to Cln3) and the six B-type cyclins (Clb1 to Clb6). As the name implies, the G₁ cyclins primarily regulate events during the cell cycle interval between mitosis and DNA replication. The yeast B-type cyclins receive their name from homology to the B cyclins of metazoans (546, 610) and are expressed in three successive waves from Start to M. With the exception of CLN3, all of the cyclin genes are paired, with both members of each pair possessing a common overall amino acid sequence and a similar pattern of transcription. Each of the cyclins confers a limited range of functions on Cdc28. The ranges overlap extensively, however, and this has considerably complicated the interpretation of investigations into their function. In this section, the structural and functional properties of the nine cyclin activators of Cdc28 are summarized. The mechanisms controlling cyclin abundance are discussed in later sections.

The three G₁ cyclins constitute an essential gene family; i.e., the loss of any two CLN genes is tolerated, but at least one must be expressed or the cells arrest at Start (106, 214, 462). Despite this genetic overlap, the CLN gene products differ in their functions, properties, and regulation (an outline of the relationships among the key G₁ regulators is given in Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Simplified outline of the relationships among major cell cycle regulators during the G1-to-S transition. Arrows indicates stimulatory interaction, lines ending in a "T" indicate inhibitory interactions.

Cln1 and Cln2. CLN1 and CLN2 were originally identified as high-copy-number suppressors of cdc28-4^ts mutations (214). These cyclins are 57% identical, but the homology rises to 72% identity in their N-terminal halves, which contain the cyclin box. The more divergent C termini contain determinants that destabilize the protein by a ubiquitin-dependent mechanism (see "Start proteolysis"). Using the crystal structure of human cyclin A as a guide for an extensive mutational analysis of CLN2, Huang et al. (253) have concluded that the cyclin box domains of Cln2 and cyclin A have similar structures, with the possible exception that helix 4 is missing or is unimportant for the function of Cln2. Genetic analyses (214, 462), coimmunoprecipitation experiments (578, 617), and in vitro reconstitutions (for Cln2 only) (122) show that Cln1 and Cln2 bind to Cdc28 and activate its protein kinase activity, presumably by a mechanism similar but not identical to that seen for the activation of human Cdk2 by cyclin A. Short of a crystal structure for any of the yeast Cdc28-cyclin complexes, genetic methods are being used to probe differences in Cdc28 recognition by different cyclins. Levine et al. have isolated Cdc28-csr mutants that are defective in Cln2 binding and kinase activity but do not affect Clb2 binding and activity (328). Cln3 binding is also diminished but not as dramatically as for Cln2. These mutations, K187E and Q188P, are in the T loop and identify a potential site of Cln2-Cdc28 interaction not seen in the crystal structure of the Cdk2-cyclin A complex. Loss of the C terminus also seems to destabilize the Cln1-Cdc28 interaction (33), indicating the presence of an interaction that is also not predicted by the existing crystal structure.

cln1 cln2

Cln3. In many ways, Cln3 is the oddest of the Cdc28 cyclins. It does not have a close yeast homolog and has only ~20 to 25% identity to its namesakes, Cln1 and Cln2 (107, 396), and actually has greater overall sequence similarity to Clb5 and Clb6. Sequence similarity is highest in the cyclin box region. Activation of Cdc28 protein kinase activity probably occurs in a manner similar to activation by Cln1 and Cln2, but the strength of the cyclin-CDK interaction and specific protein-protein contacts no doubt differ. Cross and Blake have isolated a mutant Cdc28, Cdc28-5r83, that binds Cln1 but not Cln3 (109), providing an entrée to the genetic analysis of differences in Cdc28 activation by the G₁ cyclins.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Birth, life, and death of Cln3. An outline of processes influencing the synthesis, activation, and destruction of Cln3 is shown. Heavy, open arrowheads indicate transitions involving CLN3 and its gene product. Lighter, solid arrowheads denote cellular components and environmental influences that positively regulate the indicates tep. T-shaped lines denote cellular components and environmental influences that negatively regulate the indicated step. The circled "P" indicates a phosphorylated protein, "ubi-" indicates a ubiquitinated protein. Indicated relationships may be indirect, and some steps are speculative. See the text for details.

Clb5 and Clb6. Clb5 and Clb6 are 50% identical. Of the six Clb proteins, these two have the least similarity to metazoan B-type cyclins, with Clb5 being more divergent than Clb6. For both proteins, the cyclin homology is in the C-terminal half of the protein. Clb5 can directly activate Cdc28 protein kinase activity in vitro (173, 519), and both Clb5 and Clb6 interact with Cdc28 in a two-hybrid assay in vivo (310). Clb5 possesses a mitotic destruction box (160, 310, 487) that may accelerate Clb5 proteolysis during mitosis (see "Anaphase proteolysis"), but Clb6 does not (310, 487). Clb5 also possesses a highly acidic domain that is not shared with Clb6.

cln1 cln2 clb5 clb6

cln1 cln2 cln3

clb5 clb6

Clb3 and Clb4. CLB3 and CLB4 were originally identified by high-copy-number suppression of the G₂-arresting cdc28-1N mutation (541), degenerate PCR (181, 460, 541), and low-stringency hybridization (460). The C-terminal 276 residues of both proteins contain the region most homologous to cyclin B and are 62% identical to each other. Destruction box consensus regions are found within the less homologous amino termini (see "Anaphase proteolysis"). Like CLB5, CLB3 has a highly acidic domain. CLB3 and CLB4 transcripts arise near the beginning of S phase (after the CLN1 and CLN2 peak) and remain high until late anaphase (160, 181, 460). The associated protein kinase activity has a similar periodicity (209). Measurements of absolute levels of protein kinase activity in asynchronous cells indicate that Clb3-Cdc28 constitutes the majority (67%) of all Cdc28 activity in asynchronous log phase cultures. Clb4-Cdc28 is a minor component. This abundance is not reflected phenotypically, though, since clb3, clb4, and clb3 clb4 mutants have no obvious mitotic phenotypes (181, 460, 487). The clb3 clb4 clb5 triple mutant, however, cannot make spindles and is inviable. The clb3 clb4 clb5 clb6 mutant, also inviable, has difficulty initiating S phase (487). Given the timing of their appearance, it appears that Clb5 and Clb6 are normally involved in S-phase initiation, although Clb3 and Clb4 can fill in if necessary. Clb3 and Clb4 appear to play a role in spindle formation that cannot be fulfilled by Clb5 and Clb6 but can be accomplished by Clb1 and Clb2, which appear later (10, 460).

Clb1 and Clb2. The CLB1 and CLB2 genes were cloned along with CLB3 and CLB4 as high-copy-number suppressors of the G₂-arresting cdc28-1N mutation (541), degenerate PCR (181, 196, 460, 541), and low-stringency hybridization (460). CLB2 and CLB5 are adjacent genes transcribed convergently. CLB1 and CLB6 are arranged similarlya fortunate circumstance that facilitated the cloning of both CLB5 and CLB6 (310, 487). This arrangement is apparently an evolutionary holdover, reflecting two successive duplications of a primordial CLB gene. There is no indication that CLB2 and CLB5 or CLB1 and CLB6 are regulated coordinately at the genetic level. The C-terminal 276 residues of both proteins contain the region most homologous to cyclin B and are 78% identical to each other (62% identical overall) but only 40 to 44% identical to the analogous region of Clb3 and Clb4. Destruction box consensus regions are found within the less homologous amino termini (see "Anaphase proteolysis"). As previously observed for cyclin B in Xenopus oocyte lysates (395), Clb2 mutants lacking the destruction box have difficulty exiting M phase (196, 540), indicating that CDK activation and inactivation are needed for proper cell cycle advancement.

clb2 clb4

clb2 clb5

clb1 clb3 clb4

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Processes centered around Clb2 activation, regulation, and destruction. Conventions and caveats are as in Fig. 2.

Cks1, the budding-yeast homolog of the Schizosaccharomyces pombe p13^Suc1 protein (60, 239), is essential for proper Cdc28 function (213), but the nature of this function has been mysterious and controversial. Cks1 binds to many, but not all, CDK-cyclin complexes with high specificity, an activity that has been exploited as a tool to purify CDKs (317). Recent biochemical data argue strongly for a role as a CDK-cyclin assembly factor (173, 519, 595), but this does not preclude additional functions for this small protein. The budding-yeast gene was originally cloned along with CLN1 and CLN2 as a high-copy-number suppressor of cdc28^ts mutants (213). It is highly conserved but has an extended C-terminal tail containing a 16-residue polyglutamine tract not found in its human or fission yeast counterparts (213, 461). Cks1 abundance does not vary with the cell cycle (213). Mutants lacking CKS1 arrest at Start (213), at G₂, or in a mixture of G₁ and G₂ states (549) depending upon how Cks1 function is eliminated. Studies in other systems indicate that at least part of the essential function of Cks1 is its interaction with a CDK, since mutations in either CKS1 (606) or CDC28 (143) homologs that reduce Cks1-Cdk binding are lethal. Cks1 is not needed for CDK catalytic function per se, however, since cks1^ts cells at the restrictive temperature possess high levels of Cdc28 protein kinase activity (549) and purified human Cdc2-cyclin B complexes lacking the human Cks1 homolog retain full protein kinase activity (316). In vitro, Cks1 is required to reconstitute active Cdc28-Cln2 (173, 519, 595) but is not needed for Cdc28-Clb5 activity (173), supporting a role for Cks1 as an assembly factor in vivo for at least some CDK-cyclin complexes. If this is the only role of Cks1, the G₂ arrest phenotype of cks1 (549) predicts that the M-phase Cdc28 complexes may also require Cks1 for their assembly. A test of this hypothesis has not yet been reported.

Overexpression of Cks1 delays G₂ progression (461), indicating that Cks1 may do more than simply promote Cdc28-cyclin assembly. Studies on Cks1 homologs in other systems have suggested other potential functions, including narrowing of the CDK substrate specificity (316), inhibition of CDK dephosphorylation on phosphotyrosine (see "Phosphorylation of Cdc28") (145, 429), inhibition of CDK activation following phosphotyrosine hydrolysis (265), and inhibition of CDK activity following mitosis (387, 429). Compensating for the lack of hard information on Cks1 function, there is abundant structural data on the Cks1 protein. The crystal structures of the S. pombe p13^Suc1 homolog (50, 157), the human CksHs1 (24) and CksHs2 (425) homologs, and the human CksHs1-Cdk2 complex (51) have been solved. The free Cks1 can undergo dramatic conformational changes and exists as monomers, dimers, or hexamers. Only the monomer is capable of binding CDKs, however (606), and the relevance of the multimeric forms is not clear. Watson et al. have proposed that regulated oligomerization of Cks1 may control its association with Cdk complexes (606). The crystal structure has also revealed the presence of an "anion-binding site" capable of interacting with phosphate and sulfate (50, 157, 425) that might target Cdc2 complexes to other phosphoproteins (51, 429). Sudakin et al. suggest that one such target is the APC, the complex responsible for ubiquitinating A and B cyclins at anaphase (see "Anaphase Proteolysis") (536). The phosphorylated APC binds to Cks proteins, most probably through the anion binding site, and these investigators have speculated that this might be important for Cdk-cyclin B degradation at anaphase.

CDC28 INHIBITORS: CKIS

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Far1

FAR1 was originally discovered as a gene required for mating-pheromone-induced cell cycle arrest but not needed for induction of pheromone-responsive genes (83). The gene product was initially reported to be an inhibitor of Cln1-Cdc28 and Cln2-Cdc28 protein kinase activity (436, 577) and later to have activity against Cln3-Cdc28 complexes as well (264), but it was not able to inhibit Clb5-Cdc28 and Clb2-Cdc28 in vitro (436). The biochemical nature of Far1 activity has recently been called into question by Gartner et al., who found that Far1 did not reduce the specific activity of immunoprecipitated Cln2-Cdc28 from mating-pheromone-treated cells although Far1 was present in the Cln2-Cdc28 immunoprecipitate (191). Gartner et al. have argued that the previous results may be an artifact of overproduction of Far1, Cln2, or both, but they did not provide data that supported an alternative mechanism for Far1 action. These newer results are difficult to reconcile with the previous findings in this field and indicate that much of the biochemistry in this area may need to be reevaluated. In this review, we will still consider Far1 to be a specific inhibitor of Cln-Cdc28 complexes with the caveat that its substrate specificity and possibly its mechanism of action may undergo considerable revision in the near future.

If the traditional mode of action for Far1 is upheld, Far1 probably inhibits by substrate exclusion, since Cln-Cdc28 activity is regained when Far1 is washed off the complexes (ruling out irreversible modification or disruption) (436) and since Far1 can be phosphorylated in Cln-Cdc28-Far1 complexes (making allosteric change to an inactive form of Cdc28 unlikely) (577). The Cln-Cdc28 binding and inhibitory activity has been mapped to residues 99 to 390. (Note that the original sequence analysis missed the first 150 bases of the coding sequence [376]). The positions in this review have been corrected for that difference.) The sequence of this region does not show any homology to other CKIs. The N terminus confers regulated instability on Far1 (376) (see "Start Proteolysis"), and the C terminus plays a separate role, not yet related to Cdc28 regulation, in mating and bud site selection (83, 87, 139, 590), which is not discussed further in this review.

Far1 is regulated at multiple levels. Its transcription is cell cycle regulated, with a peak near the M/G₁ transition (375) (see "M/early-G₁-specific transcription"), suggesting that Far1 may have a cell cycle function independent of its role in mating. Consistent with this, Far1 is found bound to Cln1-Cdc28 and Cln2-Cdc28 complexes in cells unexposed to pheromone and far1 strains have a reduced G₁ phase relative to the wild type (376), indicating that Far1 acts constitutively to moderate Cln activity at Start. FAR1 is not expressed in diploids and is presumably under Mata-Mat repression (83). Mating pheromone induces additional Far1 transcription (83), and this induction is necessary but not sufficient for pheromone-induced cell cycle arrest (84, 375). The protein product is predominantly nuclear (as a green fluorescence protein fusion) (235). It is stable in G₁ but is degraded rapidly following Start (375) (see "Start Proteolysis").

Far1 is unique among the known CKIs, in that its inhibitory activity is apparently enhanced by an inducible, posttranslational modification. Activation of the pheromone response pathway stimulates increased association of Far1 with all three Cln-Cdc28 complexes (435, 577). This is not simply due to increased FAR1 expression, since overexpression of FAR1 in the absence of pheromone does not stop cell cycle progress and has only weak effects in a cln1 CLN2 cln3 strain (84, 375, 413). Furthermore, wild-type Far1 from cells not treated with pheromone or produced from bacterial expression systems apparently has little or no inhibitory activity in vitro, although Far1 from mating-pheromone-treated cells or a constitutive Far1 allele (Far1-22^S87P) produced in bacteria is fully active (436). Attention has focused on phosphorylation by Fus3, a mitogen-activated protein (MAP) kinase homolog, as the required activator of Far1 because (i) Fus3 is at the base of the protein kinase cascade that responds to mating pheromone; (ii) Fus3 interacts with Far1 in a two-hybrid assay; (iii) overexpression of FAR1 suppresses the sterility of fus3^ts mutants, but FUS3 overproduction does not suppress far1 mutations; (iv) Fus3 isolated from pheromone-treated but not untreated cells phosphorylates Far1 in vitro; (v) phosphorylation of Far1 is lost in any mutation which inactivates the pheromone response pathway; (vi) mutants of Fus3 which are defective in cell cycle arrest but not mating-inducible transcription do not phosphorylate Far1; and (vii) Far1 associates with Cln-Cdc28 complexes in pheromone-treated FUS3⁺ but not fus3 cells (154, 164, 435, 577). Despite this evidence, there has been no report of Fus3 activation of Far1 inhibitory activity in vitro. The threonine at position 306 may be the phosphorylation site for whatever kinase, probably Fus3, is critical to Far1 activation, since the Far1-T306A mutant protein lacks the pheromone-induced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility shift seen with wild type Far1, does not bind to Cln2-Cdc28 complexes, and is inactive in effecting a pheromone-mediated cell cycle arrest (191).

The inability to reconstitute an in vitro assay of Cln-Cdc28 inhibition by wild-type Far1 has complicated the determination of the substrate specificity of Far1. The original genetic analysis was interpreted to suggest that Far1 would be active only against Cln2-Cdc28 complexes (83). This rested primarily on the observation that while far1 mutants were unable to stop dividing in the presence of mating pheromone, double mutants combining far1 with cln2 were pheromone sensitive. Double or triple mutants combining far1 with cln1, cln3, or cln1 and cln3 are pheromone resistant (83). The implication of this result is that Far1 is the only activity holding Cln2-Cdc28 in check and that other mechanisms would be needed to inhibit Cln1-Cdc28 and Cln3-Cdc28, but it does not prevent Far1 from having activity on Cln1-Cdc28 or Cln3-Cdc28. In accord with a broader specificity for Far1, Tyers and Futcher found that Far1 binds all three Cln-Cdc28 complexes (577). After mating-pheromone addition, the binding to Cln1-Cdc28 and Cln2-Cdc28 rises within minutes from an already appreciable basal rate. In contrast, the binding to Cln3-Cdc28 is not detectable until after an hour of pheromone exposure. The binding correlated with partial inhibition of Cln1-Cdc28 and Cln2-Cdc28 activity, but changes in Cln3-Cdc28 activity, which are difficult to measure, were not detected in this study. Using a different approach to enhance Cln3-Cdc28 activity (Cln3 overproduction instead of inhibition of Cdc34-dependent ubiquitination) Jeoung et al. have found that Cln3-Cdc28 activity is down-regulated 10-fold in a FAR1- and mating-pheromone pathway-dependent manner (264). The time course of inhibition of Cln3-Cdc28 after pheromone addition was considerably longer than that seen for Cln1-Cdc28 and Cln2-Cdc28occurring on the order of a cell cycle instead of in minutesin accord with the kinetics of Far1 binding (577), suggesting that a cell cycle-regulated process was necessary to activate Far1 for Cln3-Cdc28 inhibition. Supporting this conjecture, Cln3-Cdc28 activity was not inhibited by pheromone addition in cells blocked in G₂/M by nocodazole (264). The biochemical nature of this cell cycle-dependent activation is not clear, but stabilization and overexpression of Far1 restores Cln3-Cdc28 inhibition in G₂/M, indicating a potential role for proteolysis. The putative inhibition of Cln3-Cdc28 complexes by Far1 is generally very sensitive to the relative levels of Cln3 and Far1. Mutations or genetic constructs that increase Cln3 production or stability are mating-pheromone resistant (112, 131, 214, 235, 264, 396, 578), a resistance that can be counteracted by overproduction or stabilization of Far1 (235, 264). Similar overproduction or stability of Cln2 or Cln1 produces cells that are considerably less resistant to mating pheromone, which is more readily reversed by higher Far1 levels (112, 214, 235, 436). The data give the impression that Far1 is a potent and rapidly acting inhibitor of Cln1-Cdc28 and Cln2-Cdc28 complexes, but is less potent and acts more slowly against Cln3-Cdc28. This impression will have to be reevaluated in the light of the data of Gartner et al. (191).

Peter and Herskowitz have shown that a bacterially produced Far1 mutant, Far1-22^S87P, inhibits Cln1-Cdc28 and Cln2-Cdc28 complexes in vitro (436). This mutation eliminates a Cdc28 phosphorylation site and results in a hyperstable gene product in vivo (see "Start proteolysis") (235). Expression of FAR1-22^S87P results in constitutive, pheromone-independent cell cycle arrest and seems to cause arrest in cell cycle intervals past Start. Truncations that remove the first 50 amino acids of Far1 also hyperstabilize the protein and, when expressed in yeast, can cause cells to arrest (still in a pheromone-dependent manner) in a budded state (376). These results open up the possibility that hyperstabilized Far1 has additional targets that Far1⁺ does not. These targets are apparently not Clb5-Cdc28 and Clb2-Cdc28 if the in vitro assays are reliable indicators of in vivo function (436).

Sic1 is an inhibitor of Cdc28-Clb complexes (380, 486) and thus has an activity complementary to that of Far1. Originally discovered as a tight-binding Cdc28 substrate in immunoprecipitated Cdc28 complexes (457), it was later shown to have CDK-inhibitory activity (380), the first biochemical demonstration of CKI activity. Like Far1, Sic1 inhibitory activity is due to its ability to exclude substrates from the Cdc28 active site. Cdc28-Clb5 binding activity has been mapped to the C-terminal half of Sic1 (596). This domain has weak similarity to the inhibitory domain of Rum1, an S. pombe CKI that has many functional and regulatory parallels with Sic1 (476). There is no noticeable resemblance to mammalian CKIs, but two sequences at the extreme C terminus of Sic1 (but not found in Rum1) match the ZRXL motif that has been proposed to be a CDK-cyclin recognition motif (1). Sic1 protein expression is limited to the G₁ phase (137, 382). This pattern of expression is due to periodic transcription peaking at the G₁/M-phase border (see "M/early-G₁-specific transcription") and to Cln-Cdc28-dependent proteolysis at Start (see "Start proteolysis"). An N-terminal domain is sufficient and necessary for Sic1 ubiquitination in vitro (596) and thus may regulate Sic1 stability in vivo.

SIC1 is a nonessential gene (137, 409), but sic1 cultures contain a high percentage of cells permanently arrested in G₂ (409). Two major functions have been assigned to Sic1, and either function could account for the dying cells. The first is to prevent premature S-phase initiation until after Cln-Cdc28 levels have risen sufficiently to complete bud initiation and spindle pole body duplication (486). This function is carried out by inhibiting Clb5-Cdc28 and Clb6-Cdc28 complexes until Sic1 is destroyed, which is, in turn, initiated by Cln-Cdc28-dependent phosphorylation of Sic1 (see "Start proteolysis"). Sic1 destruction is the only essential function of the CLN genes, i.e., a cln1 cln2 cln3 sic1 mutant is viable (161, 480, 576). The second major function is to assist in the down-regulation of Clb-Cdc28 activity in late anaphase to telophase (137, 571). The exact role of Sic1 in this process is not clear; it may inhibit a fraction of Clb-Cdc28 activity that is not accessible to proteolysis at anaphase, or it may help down-regulate Clb-Cdc28 so that anaphase proteolysis can be activated (see "Anaphase proteolysis") (7).

Cdc6 also has characteristics that indicate that it may act as a Cdc28 CKI. It is better known for its role in ensuring single rounds of DNA replication during a cell cycle (reviewed in reference 566). In addition to or as part of this function, Elsasser et al. have reported that Cdc6 can bind and inhibit Clb-Cdc28 complexes (156), a function that may explain the G₂ delay seen in Cdc6 overexpressers (34, 68). This inhibitory activity of Cdc6 is enhanced by Clb5 and Clb6 by both transcriptional and posttranscriptional means (34). The Cdc6-Clb-Cdc28 interaction appears weaker than the Sic1-Clb-Cdc28 interaction, since Sic1 can displace Cdc6 from Clb-Cdc28 complexes (156). Like Sic1, Cdc6 is an unstable protein and is destroyed at the G₁/S border (438). Its destruction is dependent upon the same ubiquitination system that degrades Sic1 (see "Start proteolysis") (141, 437), but the role of phosphorylation in initiating Cdc6 degradation has not been established. Cdc6 is phosphorylated by Clb-Cdc28 complexes (156, 437), not Cln-Cdc28 complexes (156), so if Cdc28-dependent phosphorylation is a required prerequisite for Cdc6 proteolysis, Sic1 destruction would precede Cdc6 turnover. CKI activity by Cdc6 may be a means of fine-tuning Clb5 and Clb6 activity in a highly localized manner at the origin of replication.

PHOSPHORYLATION OF CDC28

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Stimulatory Phosphorylation on T169

The major site of phosphorylation on Cdc28 is at T169 (93). Consistent with the requirement for phosphorylation at the equivalent position in the CDKs of other organisms, the nonphosphorylatable Cdc28-T169A mutant cannot be activated in vitro (122) or support cell division in vivo (342). Three groups independently identified the S. cerevisiae CAK that phosphorylates T169 by purifying Cdc28-activating activity (one group finding that yeast CAK was stably associated with Cdc28 [560]) and cloning its gene, CAK1 (165, 275, 560). CAK1 has also been identified by its synthetic lethality with the sit4 protein phosphatase (543) and the kin28 CDK (560), as a suppressor of the sporulation defect of the smk1 MAP kinase (600), and as a mutant that gives a cdc34-like phenotype (91). Sequence comparisons indicate that, within the protein kinase family, Cak1 is most closely related to the CDKs but is a very distant relative. It has some unusual sequence features, the most striking being the lack of the almost invariant GxGxxG motif involved in nucleotide binding in most other protein kinases (Drosophila NinaC and Yersinia YPKA are other exceptions [91]). In addition, a mutant allele of Cak1 in which arginine replaces an invariant lysine (K31R) thought to be involved in phosphate transfer during catalysis can still support vegetative growth (600). This mutation would cripple most other protein kinases. Also, unexpectedly for a CDK-activating kinase, Cak1 is clearly active as a monomeric protein; i.e., no cyclin subunit is required for its activity (165, 275, 560).

Both monomeric CDKs and CDK-cyclin complexes act as substrates for Cak1, although only the CDK-cyclin complexes gain kinase activity upon phosphorylation by Cak1 (165, 275, 560). Cdc28 activation by Cak1 is essential for viability, and, accordingly, CAK1 is an essential gene. Immunodepletion (165, 275) and assays of extracts from cak^ts mutants (560) indicate that Cak1 is the predominant, if not the only, protein kinase capable of phosphorylating and activating Cdc28. Cdc28 activation is also the only essential function of Cak1. Cross and Levine have identified mutant derivatives of CDC28-T169E that no longer require Cak1 for activation. In these multiply mutant CDC28 backgrounds, CAK1 is no longer essential but the cak1 derivative had a slow-growth phenotype relative to the CAK1⁺ control, indicative of another, nonessential function (111). This other role has not been identified. Unlike CAKs from other species, Cak1 is unable to phosphorylate the carboxy-terminal repeat domain of RNA polymerase II (275). At least one cak1 allele (civ1-4) is synthetically lethal with kin28^ts mutations and CAK1 overexpression suppresses kin28^ts alleles (560), opening the possibility that Cak1 acts on Kin28 and possibly other yeast CDKs in addition to Cdc28. Sutton and Freiman, however, saw no effect on generalized transcription or acid phosphatase secretion in a cak1-22 strain at the restrictive temperature, as would have been expected if Kin28 or Pho85 activity were inhibited (543). Cak1 is required for spore wall morphogenesis during the later stages of meiosis (600), but this defect is not expected to lead to mitotic growth defects.

CAK1 mutant phenotypes are complex. Temperature-sensitive alleles of cak1 arrest cell division at multiple stages, with the fraction of cells found at each particular stage being determined by the cak1 allele (91, 165, 275, 543, 560, 600). The civ1-4 allele of CAK1 isolated by Thuret et al. (560) arrests predominantly in G₁, a C-terminal truncation isolated by Chun and Goebl (91) has a cdc34-like phenotype (pre-S arrest with multiple buds), and the cak1-1 and cak1-22 alleles of Kaldis et al. (275) and Sutton and Freiman (543) arrest predominantly in G₂. These varied phenotypes are consistent with a failure to fully activate Cdc28 protein kinase activity, with individual Cdc28-cyclin complexes being differentially affected in an allele-specific manner. As a consequence, CAK1 genetic interactions are also complex. The cak1-1 allele of Kaldis et al. is suppressed by overexpression of CLB2, but not CLN2, and is synthetically lethal with clb2 and with a cln1 cln3 double mutation (275, 543).

Do cyclical changes in the T169 phosphorylation state of Cdc28 play a role in cell cycle entry or progression? There is evidence that dephosphorylation of T169 plays a role in anaphase in Xenopus (349). In an attempt to simulate the constitutive phosphorylation of T169 in S. cerevisiae, Lim et al. (342) replaced T169 with glutamate. The Cdc28-T169E protein has weak but noticeable kinase activity relative to the wild type, and its expression allows the growth of cdc28-1N (mitosis-defective) but not cdc28-4 (Start-defective) mutant strains. Clb2 coimmunoprecipitates with Cdc28-T169E, but Cln2 does not. The multiply mutant, Cak1-independent Cdc28-T169E derivatives constructed by Cross and Levine promote almost normal growth and behavior even when complementing a cdc28 deletion, but, despite binding Cln2 more efficiently, they also had a severe defect in Cln2-associated protein kinase activity measured in vitro (111). These results suggest that G₁ cyclins might be more efficient activators of unphosphorylated Cdc28, while the B-type cyclins require phosphorylated T169 to be fully active. In any case, there does not appear to be a strong requirement for periodic phosphorylation/dephosphorylation of Cdc28 T169 in the usual laboratory physiological tests.

There is also no indication that Cdc28 T169 phosphorylation is periodic. There is little bulk change in the Cdc28 phosphorylation state in response to G₁ arrest by starvation or mating-pheromone exposure (212) or during the cell cycle (9), but these studies would have missed changes that affected only the small fraction of Cdc28 bound to a particular Cln or Clb (615, 617). Cak1 activity appears to be constitutive throughout the cell cycle (165, 543), but studies on its regulation are still at an early stage. Cak1 autophosphorylates (543), but the significance of this activity is unclear since the autophosphorylation is severely reduced in the cak1-K31R allele (543), which has an otherwise wild-type phenotype (543, 600). Proteins appear to associate with Cak1 in immunoprecipitates in substoichiometric amounts, but their identities or functions are unknown (543). The flip side of the coin, a protein phosphatase analogous to the mammalian KAP (CDK-associated phosphatase) (443) that would dephosphorylate phospho-T169, has not been identified. Sit4 is a potential candidate, since sit4 alleles are defective in cell cycle events associated with G₁ CDK activation (175), but the synthetic lethality of sit4 cak1 strains is difficult to explain in this context (543).

Y19. Y19 is clearly phosphorylated in Cdc28, reaching maximal levels in S and G₂ and being undetectable in M and G₁ (9, 525). Different groups using different approaches have reported widely varying extents of Cdc28 inactivation by Y19 phosphorylation, ranging from inconsequential to substantial (47, 331, 341, 344, 525). Despite these uncertainties, it is generally thought that tyrosine phosphorylation of Cdc28 delays entry into mitosis. Consistent with this and indicative of a specific effect at mitosis, Cdc28-Y19E (which partially mimics the permanently phosphorylated form) retains near-wild-type protein kinase activity in anti-Clb2 immunoprecipitates and supports DNA replication, bud emergence, and spindle pole body duplication but does not support spindle pole body separation or nuclear division (341). Phosphorylation on Y19 increases when cells are UV irradiated (195) or arrested by hydroxyurea (9), consistent with a role for Y19 phosphorylation in an S-phase or DNA damage checkpoint. Surprisingly, however, cells expressing the unphosphorylatable CDC28-Y19F allele do not differ from the wild type with respect to arrest due to DNA damage or incomplete DNA replication (9, 525), precluding an essential role in the checkpoint coupling DNA metabolism with mitosis. On the other hand, Y19 phosphorylation is an essential part of a different checkpoint system used to delay nuclear division when bud formation (polarized growth) is prevented or delayed (331). In addition, Y19 phosphorylation may play a role in mitotic exit (see "Cdc20, Cdh1, and regulation of anaphase proteolysis") (384, 605).

Swe1. Despite monitoring a different cell cycle checkpoint, the proximal regulators of Cdc28 Y19 phosphorylation are homologous to those originally described in S. pombe. SWE1, which phosphorylates Y19, encodes a protein kinase homologous to S. pombe wee1 (47, 471). The action of Swe1 is cyclin specific, since it phosphorylates Cdc28-Clb2 but not Cdc28-Cln2 or Cdc28-Cln3 (47). The basis for this specificity has not been determined but may reside in a recognition site in Clb2, a conformational change in Cdc28 induced by Clb2 but not Cln cyclins, or the occlusion of a recognition site on Cdc28 by the Cln cyclins but not by Clb2.