Introduction

The eukaryotic cell division cycle is controlled via the sequential activation and inactivation of cyclin-dependent protein kinases (cdks). In the budding yeast Saccharomyces cerevisiae, Cdc28p is the only CDK involved in regulating the cell cycle, while in higher eukaryotes, multiple CDKs (CDC2 [CDK1], CDK2, CDK4, and CDK6) control cell cycle progression. CDK substrates are assumed to be key players in most major cell cycle events. To ensure the proper timing and coordination of cell cycle events, intracellular and extracellular signals modulate CDK activities through a variety of mechanisms, including association with regulatory subunits (cyclins, inhibitors, and assembly factors), subcellular localization, transcriptional regulation, selective proteolysis, and reversible protein phosphorylation.^1–7

Binding of cyclins to CDKs is required for kinase activity. Cyclins also likely contribute to the substrate specificity^8,9 and to the subcellular localization of CDKs.^10–14 Furthermore, cyclin binding leads to multiple phosphorylations on the CDK in Xenopus egg extracts.^15,16 Cyclins are synthesized and degraded periodically during the cell cycle.^1,4,17,18 The synthesis of cyclins is mostly controlled at the level of transcription,¹⁹ and they are degraded by ubiquitin-mediated proteolysis. Cyclins are recognized by the ubiquitin-protein ligases APC (anaphase-promoting complex) or SCF (Skp1p-Cdc53p/cullin-F-box protein), polyubiquitinated, and destroyed by the 26S proteasome.^20,21 In the budding yeast, nine cyclins form complexes with Cdc28p: three G1 cyclins (Cln1p to Cln3p) and six B-type cyclins (Clb1p to Clb6p). In the G1 phase, Cln1-3p regulate cell cycle events such as “Start” (equivalent of restriction point in mammalian cells),^22,23 the repression of pheromone-induced transcription, and meiosis in diploid cells.^24,25 B-type cyclins are expressed in three successive waves from Start to mitosis and regulate most events during the cell cycle (reviewed in 26).

In budding yeast, there are two Cdc28p inhibitors (CKIs): Far1p and Sic1p. Far1p, which is required for pheromone-induced cell cycle arrest,²⁷ inhibits the kinase activities of Cln1p-Cdc28p, Cln2p-Cdc28p, and Cln3p-Cdc28p,^22,28,29 but it is incapable of reducing Clb5p- or Clb2p-associated Cdc28p activities.²⁹ In contrast to Far1p, Sic1p is a specific inhibitor of B-type cyclin-Cdc28p complexes.^30,31 Sic1p has two functions: in G1, it prevents premature S-phase entry before bud initiation and spindle pole body (SPD) duplication.³¹ At the end of mitosis, it promotes mitotic exit by inhibiting Clb-Cdc28p activity.^32,33 It should be noted that there is no apparent sequence homology between yeast CKIs and their mammalian counterparts. Mammalian CKIs are classified in two groups: the CIP/KIP family consisting of p21^{CIP1/WAF1,34–36} p27^KIP1,^37–39 and p57^KIP240,41 which inhibit all CDKs; and the INK4 family including p15^INK4b,42 p16^INK4a,⁴³ p18^INK4c,⁴⁴ and p19^INK4d45,46 that are specific CDK4/CDK6 inhibitors.

In addition to binding of protein subunits, CDKs are regulated by protein phosphorylation (for review see 7). There are two inhibitory and one activating phosphorylation sites. The inhibitory phosphorylations (which occur on Thr-14 and Tyr-15 in human CDC2) are carried out by the WEE1-like protein kinases (Swe1p in budding yeast) and removed by the CDC25 phosphatase family (Mih1p in budding yeast) (for reviews see 3, 7). In higher eukaryotes, inhibitory phosphorylation regulates timing of entry into M phase.⁴⁷ In Saccharomyces cerevisiae, the inhibitory phosphorylation of Cdc28p on Tyr-19 by Swe1p does not play a significant role during a normal cell cycle or after DNA damage^48,49 but is required for a morphologic checkpoint that monitors the coordination of the budding and nuclear division cycles.⁵⁰ Activating phosphorylation occurs within the so-called T-loop^7,51 on a conserved threonine residue corresponding to Thr-169 in Cdc28p and Thr-161 in human CDC2. The inhibitory phosphorylations are dominant over the activating phosphorylation. Full activation of CDKs, which is necessary for normal cell cycle progression, requires binding of a cyclin, removal of inhibitory phosphorylations, and the presence of an activating phosphorylation. The latter is required for CDK activities in vitro and in vivo^16,52–57 and is catalyzed by the CDK-activating kinase (CAK).⁵⁸ In higher eukaryotes, CAK was identified as the CDK7/cyclinH/MAT1 complex (see Chapter 2). In this review, we will focus on the regulation of Cdc28p and other yeast CDKs by Cak1p, the budding yeast Saccharomyces cerevisiae CAK and by the protein phosphatase 2C (PP2C).

Historical View of Cak1p Identification

After CAK activity was detected in cell extracts, it was purified from Xenopus and starfish egg extracts and later from human and mouse cell extracts (see Chapter 2). However, determination of the physiological role of CAK is difficult in mammalian cells due to technical limitations. Therefore, it was of great interest to identify CAK in a genetically tractable system like the budding yeast. Three groups, including our own, succeeded in isolating the protein responsible for CAK in budding yeast by biochemical methods. An assumption that we and others made in these studies was that the CAK activity in budding yeast extracts would be able to activate recombinant human CDK2/cyclin complexes. It is worth noting that none of the groups identifying Cak1p used Cdc28p as substrate since it is difficult to express, it tends to aggregate, and displays erratic low activity. Human CDK2 was the preferred assay substrate in these studies since it can be easily expressed and purified. Furthermore, CDK2 has been shown to complement Cdc28p mutations,⁵⁹ indicating that it can be a Cak1p substrate in vivo. In the CAK activity assays, inactive CDK2/cyclin complexes were incubated with yeast cell extracts, thereby CDK2 could be activated and its ability to phosphorylate histone H1 was assayed. Otherwise, the assay was similar to one described earlier for studying CAK from frog and human extracts.⁵⁶

In mammals, CDK7 was isolated as an enzyme with CAK activity (see Chapter 2) and was later shown to be a subunit of TFIIH (see Chapter 8). The closest homolog to CDK7 in the budding yeast is Kin28p. Kin28p was originally cloned as a Cdc28p-like gene⁶⁰ that has a cyclin-like regulatory subunit, Ccl1p.⁶¹ The only function known for Kin28p was that it affected the transcription of some genes.^62,63 The correlation to CDK7 became almost perfect when Kin28p was shown to be a subunit of yeast TFIIH.⁶⁴ Surprisingly, Kin28p does not display any CAK activity nor does it affect the phosphorylation state of Cdc28p.^62,64 Because of the discrepancy between CDK7 and Kin28p, it was worthwhile to purify the protein responsible for CAK activity in budding yeast.

Purification of the CDK2 activating activity led to the identification of a 43-kDa protein which was named Cak1p^52,54 or Civ1p.⁵⁷ The successful purification of Cak1p depended on several critical factors including the activity assay and the ability to prepare large amounts of biochemically active yeast lysates. The source of budding yeast was another major concern in these studies. One group grew large amounts of yeast in a fermenter and lysed them by mechanical disruption [“bead beating”].⁵² Our group used freeze-dried yeast (Sigma #YCS-2) because these lysates displayed robust CAK activity. In addition, we lysed the yeast by grinding them under liquid nitrogen, which allows the preparation of large amounts of lysate.⁵⁴ In the end, both groups purified the same protein, confirming the suitability of both approaches.^52,54

Thuret et al.⁵⁷ used a different biochemical approach involving the isolation of Cdc28p from yeast extracts. They realized that the addition of cyclin A readily activated Cdc28p and hypothesized that CAK was part of the purified Cdc28p complex. After gel filtration of Cdc28p complexes, they found a CAK activity peak at 45 kDa and identified it as Cak1p (also called Civ1p) using a candidate-gene approach. They also found Cak1p in Cdc28p immunoprecipitates. Nevertheless, it is curious that such a Cak1p/Cdc28p complex of 80 kDa or more has never been observed after gel filtration,^52,54,57 and Cdc28p does not interact with Cak1p in a two-hybrid screen.⁶⁵

Four different genetic screens yielded the CAK1 gene, which was called at the time CAK1,⁶⁶ PDL3,⁶⁷ MCA28^63,68 or MEB1⁶⁹ [see below]. The first screen involved the protein phosphatase Sit4p that is required for progression from G1 to S-phase.^70,71 In the strain background used in these studies, absence of Sit4p causes a slow growth phenotype. CAK1 (formerly known as PDL3) was identified as being synthetically lethal with sit4 mutations.⁶⁷ The relation between CAK1 and SIT4 is unclear and might be indirect. The second screen used a yeast haploid-lethal mutant collection that was examined microscopically to identify mutants arrested with a morphology similar to that caused by mutation in CDC4, CDC34, or CDC53, genes that are subunits of the SCF complex.⁶⁹ One mutant was found to be identical to CAK1. Again the relation between CAK1 and CDC4, CDC34, CDC53 is not obvious, except that all these genes regulate the activity of Cdc28p through degradation of cyclins and inhibitors. In the third screen, CAK1 was isolated as a dosage suppressor of the sporulation defect of a smk12 conditional strain.⁶⁶ SMK1 is a MAP kinase involved in spore wall morphogenesis during meiosis in yeast. The functions of CAK1 in meiosis and its activity in the Smk1 pathway are discussed in further detail below (see “Cak1p and its role in meiotic development.”). In the fourth screen, Cak1p (formerly named MCA28) was identified as a dosage-dependent suppressor of kin28-ts mutations.^63,68 Kin28p is a subunit of transcription factor IIH, where it phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II^63,64 [for more details on Kin28p, see section 3.3].

Interestingly, none of the genetic screens revealed the function of CAK1 in Cdc28p activation. The reason for this might be that CAK1 is involved in all phases of the cell cycle and therefore genetically interacts with many gene products, leading to a complex mutant phenotype from which it is difficult to infer Cak1's function. A reverse approach like biochemical purification which followed function to sequence proved most successful in identifying Cak1p and its roles in the yeast cell cycle.

Although CDK7 homologs have been identified in many species from fission yeast to humans, only a few potential Cak1p-like enzymes have been identified. Csk1 of S. pombe, Cak1At of Arabidopsis thaliana, and Cak1p of Candida albicans have each been shown to rescue a cak1 mutant in S. cerevisiae.^72–74 Cak1At is closer in primary amino acid sequence to CDK7, whereas the Csk1 sequence resembles that of Cak1p. Studies in fission yeast indicate that Csk1 indeed acts as a monomer and can phosphorylate the CAK Mcs6 in vivo and cdc2 in vitro, suggesting that is has CAKAK (CAK activating kinase) as well as CAK activity.^73,75 Nevertheless, in vivo Csk1 only activates Mcs6 but not cdc2 [for details, Chapter 4].⁷⁶ Whereas CDK7 possesses both CAK and CTD kinase activities, neither Csk1 nor Cak1At have been found to phosphorylate the CTD.^73,74 We have indications that Cak1At, Csk1, and C. albicans Cak1p expressed and purified in the insect cell system are functional as a monomer and prefer to phosphorylate monomeric CDKs (authors' unpublished observations). Cyclin subunits have not been described for any of these enzymes. Thus these proteins appear to follow the pattern of budding yeast Cak1p rather than the heterotrimeric CDK7 metazoan model.

Substrates and Biochemistry of Cak1p

Cak1p belongs to the family of serine/threonine protein kinases and is a distant member of the CDK superfamily.^52,54,57 Cak1p activates CDKs in vitro by phosphorylation of the conserved CDK domain known as the T-loop [also called activation segment].⁷⁷ Cak1p activates the yeast cell cycle CDK Cdc28p^52,54,57 as well as its functional homologs Xenopus Cdc2,⁷⁸ human CDK3 (P.K., unpublished data) and human CDK2.^52,54,57 Cak1p also phosphorylates CDK6 in its monomeric as well as cyclin D1-, D2-, or D3-bound forms.⁷⁷ The metazoan CAK CDK7/cyclin H phosphorylates CDC2, CDK2, CDK3, CDK4, and the CDK6/cyclin D3 complex but not CDK6/cyclin D1/2 complexes,⁷⁷ a distinction in substrate utilization that opens the possibility for the existence of CDK-activating kinases tailored for specific CDK. Cak1p does not phosphorylate all CDKs. In the same activity assays that determined its activity on CDK6, for example, no kinase activity was observed with CDK4.⁷⁷ The K_M(CDK2) of Cak1p increases by a factor of 4 when the activating T-loop threonine is mutated to a serine [T160S].⁷⁹ Such an effect was not observed for CDK7, once again underlining the difference in the biochemistry of the two CAKs.⁷⁹ It is interesting to note that phosphorylation of CDK2 by CAK does not serve to stabilize ATP in the ATPase reaction but increases the catalytic efficiency by 100,000-fold and the turnover rate by 1,000-fold.⁸⁰

An important finding of in vitro activity assays was that Cak1p demonstrates a substrate preference for monomeric CDK rather than the cyclin-bound form.⁷⁷ This is in contrast to the mode of action of CDK7, which preferentially phosphorylates CDK/cyclin complexes.^77,81 The functional implications of this selectivity on the Cdc28p activation pathway will be discussed later in this review. It is important to note the mechanistic significance of the diverged substrate specificity between Cak1p and CDK7. Both CAKs can phosphorylate CDK2, yet binding of cyclin oppositely influences their activity, suggesting that each might recognize CDK2 in a slightly different conformation (given that cyclin binding induces conformational changes in CDK structure).

In vivo, Cak1p's primary substrate is the major cell cycle CDK Cdc28p. In fact, phosphorylation of Cdc28p at Thr-169 (the CDK T-loop site of activating phosphorylation) is the only essential function of Cak1p for normal budding yeast growth.^52,54,57 A strain that carries a mutant version of Cdc28-43244p, mimicking constitutive phosphorylation (T169E) and harboring several additional mutations in the same gene, bypasses the requirement for activating phosphorylation and is viable independently of Cak1p.⁸² Furthermore, this mutant Cdc28-43244p is capable of supporting cell cycle progression, showing that regulated reversible phosphorylation at position Thr-169 is not essential under the conditions tested. Although viable, the strain has growth defects in the absence of Cak1p, perhaps because of failure to phosphorylate other, as yet unknown, nonessential substrates of Cak1p.⁸² Conspicuous in its absence from a list of Cak1p substrates is the C-terminal domain (CTD) of RNA polymerase II's large subunit. CDK7 is a subunit of the transcription factor TFIIH complex and phosphorylates the CTD.^64,83–86 Phosphorylation of the CTD by TFIIH is thought to mediate the transition from transcription initiation to elongation. Cak1p was neither found in the TFIIH complex nor does it exhibit CTD kinase activity.^52,54,57

Such activity, however, is found in CDK7's closest S. cerevisiae relative, the cyclin-dependent kinase Kin28p,⁶² which is part of the yeast TFIIH complex.⁶⁴ Cak1p may yet maintain a connection to transcription as it was found to phosphorylate Kin28p at the conserved threonine (Thr-162) and stimulate its CTD kinase activity in vitro. Kin28p remains unphosphorylated when Cak1p is inactivated in vivo, indicating that it is a physiological Cak1p substrate.^87,88 This phosphorylation event, however, is not essential as a T162A mutant of Kin28p is viable.⁸⁸ In the absence of Thr-162 phosphorylation, Kin28p can be activated instead through binding to the assembly factor Tbf3p/Rig2p.^89,90

Biochemical Characterization of Cak1p

Cak1p is unconventional in that its primary amino acid sequence deviates in subdomains shared by the protein kinase superfamily. For example, budding yeast Cak1p entirely lacks a canonical “glycine loop” (GXGXXG) in the nucleotide-binding fold. Typically, this motif is instrumental in anchoring the nucleotide substrate, and its mutation in PKA leads to reduced catalytic activity.⁹¹ Cak1p, however, maintains reasonable affinity for ATP [K_M(ATP) = 4.8 μM, ATPase rate = 0.13 min⁻¹, compared to 17 μM and 0.66 min⁻¹ for wildtype PKA].⁹² It can also bind GTP (though with a K_M over 200-fold higher than for ATP) as well as ADP and AMPPNP.⁹² Introduction of a glycine motif by mutagenesis did not significantly affect the enzyme's catalytic rate, suggesting that other subdomains in Cak1p may be atypical.⁹² Cak1p has substitutions in highly conserved kinase core residues, and although it contains the “invariant lysine” essential for activity in most protein kinases, this residue is dispensable for Cak1p activity.⁹² In other kinases, the invariant lysine is involved in nucleotide alignment and seems to function in catalysis as its mutation has little effect on nucleotide binding but completely inhibits catalysis.⁹³ Mutations of the Cak1p Lys-31 surprisingly reduced ATP binding but left catalytic activity largely unaffected. These mutants were able to complement a cak1 deletion in vivo, suggesting that this residue is not required for activity.^66,67,69,92 Additionally, Cak1p is insensitive to 5'-fluorosulfonylbenzoyladenosine [FSBA],^77,92 which through covalent modification of the invariant lysine leads to loss of activity in nearly all kinases, including CDK7.⁵⁶ Thus, a combination of kinetic and mutagenic analysis demonstrates that Cak1p's unusual ATP binding pocket is efficiently functional. It remains to be resolved what structural adaptations provide the alternative route by which the molecule compensates for the lack of canonical protein kinase features. In an effort to further understand Cak1p biochemistry, Enke et al.⁹⁴ examined the parameters of CDK2 phosphorylation by Cak1p. It was concluded that catalysis is the rate-limiting step in CDK2 phosphorylation, which likely proceeds through a rapid-equilibrium, random pathway (where nucleotide and protein substrate binding are independent in the formation of the ternary complex). Despite sequence similarity to the CDK family, Cak1p is not cyclin dependent. Purified Cak1p is active as a monomer in vitro and is not found associated with a cyclin or any other protein in vivo as it elutes in a single peak corresponding to its molecular weight when yeast extracts are subjected to gel filtration.^52,54,57,95 Furthermore, CDKs bound to mammalian CDK inhibitors (CKI; p21^CIP1, p27^KIP1, p57^KIP2, p16^INK4a, and p18^INK4c) were phosphorylated by Cak1p but not by human CDK7.⁷⁷ Crystallographic evidence⁹⁶ suggests that CKIs inhibit CDKs by binding to the substrate, by inducing conformational changes, and potentially sterically hindering enzyme-substrate interaction. Cak1p's insensitivity to CKIs suggests that Cak1p approaches substrates in a manner that is structurally distinct from that of CDK7 or that it recognizes CDKs in a different conformation. However, we should keep in mind that also CAKs might induce a conformational change when they bind to their CDK substrate.

Regulation and Localization of Cak1p

Regulation of CDKs plays an important role in their function. Since the binding of cyclins and the activating phosphorylation are general requirements for most of the CDKs including CDK7-type CAK, it was thought that budding yeast Cak1p might also be regulated by a similar mechanism. Surprisingly, Cak1p is active without a cyclin partner and does not require post-translational modification for its full kinase activity.⁵⁴ Furthermore, Cak1p in crude yeast extract elutes as a monomer from a gel filtration column. Mutation of potential phosphorylation sites in Cak1p has no effect on its activity in vivo or in vitro.⁹⁵ In addition, isoelectric focusing of Cak1p reveals no modified species, suggesting that the majority of Cak1p is not phosphorylated.⁹⁵ The activity and protein level of Cak1p remain constant during vegetative cell growth.^52,67 In contrast, CAK1 mRNA⁶⁶ and protein levels⁹⁵ fluctuate dramatically during meiosis (discussed in the section on Cak1p and Its Roles in Meiotic Development). Although Cak1p protein levels and activity are constant, we cannot exclude that Cdc28p can only be activated at a specific time. In this model, both Cak1p and Cdc28p are present at all times, but Cdc28p can only be phosphorylated by Cak1p at a specific time. That way Cak1p would regulate Cdc28p directly. Nevertheless, we have no indication that such a model is valid since Cdc28p is fully phosphorylated at all times [see the section on The Activation Pathway of Cdc28p].⁹⁷ We cannot exclude, though, that only a small percentage of Cdc28p is phosphorylated at specific times or that this modification is only short lived. In both cases, it would be virtually impossible to detect such effects.

Whereas CDK7 is localized to the nucleus, budding yeast Cak1p is found in both the cytoplasm and the nucleus by immunofluorescence staining and is mostly found in the cytoplasmic fraction by subcellular fractionation,⁹⁵ which correlates with the localization of its major substrate, Cdc28p.⁹⁸ Furthermore, the protein levels and localization of Cak1p are constant throughout the cell cycle,^52,67 which is also consistent with the invariable phosphorylation state of Cdc28p.⁹⁷ It is unclear whether the activating phosphorylation of Cdc28p plays any regulatory role during the cell cycle in yeast.

Cak1p and Its Roles in Meiotic Development

Sporulation is the program in which diploid (MATa/MATa) yeast cells give rise to haploid spores in response to nutrient limitation (reviewed in 99). Following induction, a single round of DNA replication is followed by an elongated prophase when synapsis and genetic recombination take place. Once recombination has been completed, two rounds of chromosome segregation occur without an intervening S-phase. Homologs separate during the unique MI division, while sister chromatids separate during the mitosis-like MII division. Spore wall assembly follows the meiotic divisions. Cak1p is required for multiple processes during sporulation. Some Cak1p requirements during sporulation appear to be Cdc28p-dependent while others appear to be Cdc28p-independent. In order to review the roles of Cak1p in meiotic development it is first necessary to consider Cdc28p's role in sporulation and how the regulation of Cdc28p may differ between mitosis and meiosis.

The role of Cdc28p in meiosis was first addressed by Shuster and Byers who reported that a cdc28-4 diploid strain shifted to its restrictive temperature following meiotic induction completed chromosomal DNA replication and spindle pole body duplication and arrested prior to the nuclear divisions in pachytene [the last stage of MI prophase before cells become committed to chromosome segregation].¹⁰⁰ CDC28 is required for both DNA synthesis and spindle pole body duplication during mitosis. In seeming contradiction to the CDC28 temperature shift studies, premeiotic DNA replication does require the CLB5 and CLB6 B-type cyclins.^101,102 Taken together, these results suggest that there is a low threshold of Cdc28p catalytic activity required to complete premeiotic DNA synthesis, which is provided by the residual activity of the cdc284 allele. Another possibility that cannot be ruled out is that there is an as yet to be identified CDK that complexes with Clb5p and Clb6p and promotes DNA replication in meiosis. Premeiotic and mitotic DNA replication also differ in how CDK inhibitors are regulated. In mitosis, S-phase requires targeting of the Sic1p CDK inhibitor for destruction by Cdc28p/Cln1p and Cdc28p/Cln2p complexes.^31,103–106 In contrast, the Clns are not required for sporulation, and the destruction of Sic1p requires the sporulation-specific Ime2p protein kinase.¹⁰¹ Ime2p is not the CLB5/6 kinase that is required for DNA replication, however, since a strain lacking Sic1p can complete DNA replication in the absence of Ime2p.¹⁰¹ These results show that at least some mitotic Cdc28p functions can be performed by sporulation-specific protein kinases during sporulation.

In contrast to premeiotic DNA synthesis, there is clear evidence that Cdc28p is required for the meiotic divisions. cdc28-4 diploids sporulated at the restrictive temperature arrest in pachytene (just prior to chromosome segregation).¹⁰⁰ In addition there is good genetic evidence demonstrating a requirement for B-type cyclins in MI and II.¹⁰⁷ However, the cyclins appear to have specialized meiotic requirements. CLB1 appears to be the major Clb that controls exit from pachytene and entry into meiosis I, CLB1, CLB3, and CLB4 appear to play partially redundant roles in promoting meiosis II, and CLB2, while required for mitosis, does not appear to play any role in meiosis.¹⁰⁷

The transition from pachytene into meiosis I represents a major point of regulatory control in sporulation, and there is good evidence that much of this regulation is achieved by controlling Cdc28p activities. For example, signals that control the exit from pachytene and entry into meiosis I include the completion of genetic recombination, which is monitored by a pathway termed the pachytene checkpoint (also referred to as the recombination checkpoint). If recombination has not been completed, this checkpoint pathway blocks the program in pachytene (for review, see 108). SWE1, which encodes the kinase that phosphorylates the inhibitory tyrosine in the ATP binding pocket of Cdc28p, is required for the pachytene checkpoint, and Swe1p is activated in cells that have undergone checkpoint-mediated arrest.¹⁰⁹ An overlapping control mechanism that regulates Cdc28p activity is the transcriptional program of sporulation.^110,111 This transcriptional cascade involves the induction of several hundred promoters that can be divided into “early”, “middle” and “late” temporal classes. Early genes are expressed when premeiotic DNA synthesis, synaptonemal complex formation, and recombination are occurring. Middle genes are expressed as cells exit pachytene, enter the nuclear divisions, and begin to form spores. Late genes are expressed as spore formation is being completed. Thus, the key events of sporulation are tightly coupled to and controlled by this genetically programmed transcriptional cascade. The CLB1, CLB3-CLB6 genes are transcriptionally induced as middle genes. The sporulation-specific Ndt80 transcription factor is required for this induction.^112,113 Ndt80 is negatively regulated by the pachytene checkpoint.^112–114 In checkpoint-arrested cells, middle gene induction and the wave of CLB transcription does not occur as Ndt80 is inhibited. It is likely that the negative regulation of CLB transcriptional induction during meiosis plays a role in preventing the nuclear divisions in checkpoint-arrested cells.

Mutants in cak1 show a variety of meiotic defects. Temperature shift experiments show that CAK1 is required for the meiotic nuclear divisions⁶⁶ (E.W., unpublished data). These results are similar to those seen with conditional CDC28 mutants¹⁰⁰ and are consistent with the notion that Cak1p activates Cdc28p during meiosis. These results imply that the pool of activated Cdc28p present in prophase is insufficient to drive the nuclear divisions and raises the possibility that Cak1p can be rate limiting for the nuclear divisions in wild-type cells. Experiments to monitor the phosphorylation state of Cdc28p during meiotic development have not been reported but might help to clarify this issue.

MI and MII in yeast are rapidly followed by spore formation. This morphogenetic program initiates as a thickening of the outer plaque of the centrosome (referred to as the spindle pole body in yeast). Vesicular fusion mediated through a sporulation-specific arm of the secretory pathway is involved in the outgrowth of the prospore membrane,¹¹⁵ which surrounds the haploid meiotic products. Subsequently, spore-specific components are assembled from within and around the double-layered prospore membrane to generate the spore wall (reviewed in 99).

SMK1 encodes a sporulation-specific MAP kinase homolog that is a central regulator of spore morphogenesis.¹¹⁶ smk1 null mutants complete meiosis I and II but are defective in assembling spore walls. Moreover, different smk1 hypomorphs block the program at distinct steps in the morphogenic pathway.¹¹⁷ CAK1 in high copy number suppresses the spore morphogenesis defects of weakened smk1 mutants.⁶⁶ Furthermore, a cak1 strain that is able to complete meiosis I and II but that is specifically defective for spore morphogenesis has been isolated. In addition, CAK1 is transcriptionally induced when meiosis and spore morphogenesis are occurring. These results indicate that Cak1p is required not only for the nuclear divisions (a Cdc28p-dependent function) but also plays a role in activating the SMK1 spore morphogenesis pathway. More recently, it has been shown that Cak1p and Smk1p interact using a two-hybrid system in mitotic cells.⁶⁵ However, it has not been determined whether this interaction is direct or whether the Cak1p and Smk1p are tethered by another protein.

The expression of Cak1p protein has been examined and shown to change dramatically during sporulation.⁹⁵ During the early phase of sporulation, when DNA replication is occurring, Cak1p levels are comparable to the levels seen in mitotic cells. Subsequently, Cak1p levels fall to near background during late prophase (around pachytene). These data suggest that the level of Cak1p protein during sporulation is regulated not only at the transcriptional level but also by a developmentally regulated proteolysis pathway. Subsequently, Cak1p levels increase as middle genes are transcriptionally induced and as cells exit pachytene and enter the meiotic divisions. Since Cak1p is required for Cdc28p activity and Cdc28p is rate limiting for exit from pachytene, this might suggest that the regulation of Cak1p levels could play a role in the pachytene checkpoint. However, expression of Cak1p using a high-level promoter that generates constitutive CAK1 mRNA and protein levels does not cause bypass of the pachytene checkpoint (E.W., unpublished data).

Additional insight into the role of Cak1p in sporulation comes from the analysis of genetic backgrounds in which the mitotic role of CAK1 has been made dispensable.⁸² These studies made use of CDC28-43244, a multiply mutant form of Cdc28p, which lacks a phosphorylatable residue at position 169 (Thr-169 is the in vivo phosphorylation target of Cak1p) and additional substitutions that presumably hyperactivate the kinase. CDC28-43244 mutant cells grow in the complete absence of CAK1. While homozygous CDC28-43244 CAK1 diploids sporulate, a CDC28-43244 cak1D homozygous diploid is sporulation-defective (E.W., unpublished data). Since the Cdc28-43244 mutant protein lacks the threonine that is phosphorylated by Cak1p, these results imply that Cak1p functions in sporulation by phosphorylating a target other than Cdc28p. Biochemical studies indicate that Smk1p is inactive during sporulation of the CDC28-43244 cak1 background, suggesting that Cak1p activates a component of the SMK1 MAPK pathway (M. Shaber and E.W., unpublished data). Kin28p, the other known mitotic target of Cak1p, does not appear to function in activating Smk1p since a kin28 mutant lacking the residue normally phosphorylated by Cak1p in vegetative cells sporulates normally (J. Kimmelman and M. Solomon, personal communication).

In summary, Cak1p is highly regulated and plays multiple roles during sporulation. First it appears to be required for exit from pachytene and completion of the meiosis I and II divisions. This observation is consistent with the central role of Cdc28p in nuclear division. In addition, Cak1p appears to activate the SMK1 pathway by a Cdc28p-independent mechanism. This later sporulation-specific function has led to the suggestion that Cak1p plays a role in coordinating meiosis with the spore differentiation pathway.⁶⁶

Removal of the Activating Phosphorylation from Cdc28p

Compared to our knowledge of CAK, much less is known about the protein phosphatases that reverse the activating phosphorylation in CDKs. Previous studies in S. pombe and in Xenopus egg extracts raised the possibility that the dephosphorylation of this residue may be required for exit from mitosis^118,119 and implicated type 2A and type 1 protein phosphatases in the dephosphorylation of Cdc2.^119,120 More recently, a dual-specificity phosphatase, KAP (also called Cdi1, Cip2),^36,121,122 was shown to preferentially dephosphorylate monomeric CDK2 in vitro.¹²³ This result is consistent with the observation that Xenopus Cdc2 is dephosphorylated only after cyclin degradation.¹¹⁹ However, there is no obvious KAP homolog in budding yeast. Using Thr-169 phosphorylated Cdc28p as substrate, we biochemically identified the Cdc28p phosphatase as belonging to the type 2C family of Ser/Thr protein phosphatases (PP2C): this activity required Mg²⁺ and was insensitive to PP1/PP2A and dual-specificity/tyrosine phosphatase inhibitors such as microcystin-LR, vanadate, and tungstate.¹²⁴ Two yeast PP2Cs, Ptc2p and Ptc3p, display Cdc28p phosphatase activity in vitro and in vivo and account for ˜90% of Cdc28p phosphatase activity in yeast extracts. Overproduction of Ptc2p or Ptc3p reduces the level of Thr-169 phosphorylation in Cdc28p in vivo and results in synthetic lethality in temperature-sensitive cak1 strains at the permissive temperature. Furthermore, the dual disruption of PTC2 and PTC3 suppresses the growth defect of a cak1 mutant at semi-permissive temperature. Since the phosphorylation of Cdc28p is the only essential function of Cak1p,⁸² Ptc2p and Ptc3p are likely to be the physiological Cdc28p phosphatases in budding yeast. Like KAP, Ptc2p and Ptc3p prefer monomeric CDKs rather than cyclin-bound CDKs as substrates. PP2C-like activities are also responsible for >99% of CDK2 phosphatase activity in HeLa cell extracts.¹²⁴ We recently demonstrated that these CDK2 phosphatase activities belong to PP2Ca and PP2Cb2, the closest homologs of yeast Ptc2p and Ptc3p.¹²⁵ Therefore, the ability of PP2Cs to reverse the activating phosphorylation of CDKs is evolutionarily conserved.

The balance between activating phosphorylation and dephosphorylation of CDKs appears to vary greatly between species. For example, studies from S. pombe and Xenopus show that the activating phosphorylation of Cdc2 is removed rapidly either during or at the end of mitosis.^118,119 In budding yeast, however, Cdc28p remains phosphorylated at Thr-169 throughout the cell cycle.^3,87,97 This difference is likely due to the different substrate specificities of the respective CAKs and to the relative activities of CAK and PP2C in the different species: (i) budding yeast Cak1p can phosphorylate monomeric CDKs and Cdc28p molecules that are dephosphorylated after cyclin degradation.⁷⁷ (ii) CDK phosphatase activity in HeLa cell extracts is much higher than in budding yeast extracts. In budding yeast, dephosphorylation of Cdc28p by Ptc2p and Ptc3p happens less frequently than phosphorylation by Cak1p, as indicated by the fact that most monomeric Cdc28p molecules are phosphorylated. Using a phospho-specific antibody, we estimated that phosphorylation of Cdc28p by Cak1p is about 9-fold more frequent than dephosphorylation by Ptc2p and Ptc3p combined.⁹⁷ In contrast, PP2C-like activities in HeLa cell extract are about 10-fold higher than those in yeast extract,¹²⁴ but no comparison has been done of yeast and human CAK activities. Therefore, Cak1p plays a dominant role in determining the phosphorylation of monomeric Cdc28p in budding yeast whereas PP2Cs might play the dominant role in human cells.

The identification of the CDK phosphatases also raised two questions. First, does the removal of the activating phosphorylation have any regulatory role during the cell cycle? Second, how are these PP2Cs regulated? In budding yeast, a regulatory role for the removal of the activating phosphorylation from Cdc28p during the normal cell cycle has not been detected. Three independent lines of experiments do not currently support such a role:

1. dual deletion of PTC2 and PTC3 has no apparent phenotype,¹²⁴
2. the activating phosphorylation of Cdc28p is constant during the cell cycle, and
3. the Cdc28-43244p mutant, which mimics a permanent “phosphorylated” form of Cdc28p (T169E with several additional mutations), supports cell growth.⁸²

However, it is unclear whether the same scenario occurs in higher eukaryotes. Studies in S. pombe and Xenopus egg extracts raised the possibility that dephosphorylation at the site of the activating phosphorylation may be required for exit from mitosis.^118,119 Considering that CAK activity does not vary in cycling Xenopus egg extracts,¹⁶ it will be interesting to determine if the higher eukaryotic CDK phosphatase activity is regulated during the cell cycle. Previous studies showed that PP2Ca might be phosphorylated at its C-terminus,¹²⁶ and PP2Cb mRNA levels and activity are up-regulated during the 1α,25-dehydroxyvitamin D₃-induced monocytic differentiation of leukemic HL-60 cells.¹²⁷ Further studies will be necessary to determine whether the extent of activating phosphorylation of CDKs is modulated by the activity of PP2Cs. Like Ptc2p and Ptc3p in budding yeast, human PP2Ca and b isoforms contain potential sites for N-terminal myristoylation. It will be interesting to determine whether the CDK phosphatase is myristoylated in vivo and how the myristoylation regulates PP2C and its localization.

The Activation Pathway of Cdc28p

Because CDKs are regulated by several mechanisms, including cyclin binding, activating phosphorylation by CAKs, inhibitory phosphorylation by WEE1-like kinases, and association with inhibitory proteins, there are many possible pathways that can produce the active form of the CDK. For the budding yeast CDK, Cdc28p, the combinatorial problem is simplified somewhat because inhibitory phosphorylation does not play a major role in Cdc28p regulation during normal mitoses.^48,49 Phosphorylation of the inhibitory site, Tyr-19, is required instead for a checkpoint that monitors the coordination of the budding and nuclear division cycles (128–130). It is not known whether Tyr-19 phosphorylation in response to the checkpoint signal is dependent on prior cyclin binding or activating phosphorylation of Cdc28p; however, in other organisms, inhibitory phosphorylation occurs only after cyclin binding.

The physiological role of the Cdc28p inhibitors, Far1p and Sic1p, is to down-regulate the active Cdc28p kinase,^29,31,32,131 suggesting that they are late players in the activation pathway, associating with the CAK-phosphorylated, cyclin-bound Cdc28p complex. Both inhibitors have indeed been shown through biochemical experiments to bind and inhibit Cdc28p/cyclin complexes.^29,31

The best-studied aspects of the Cdc28p activation pathway concern the relative order of cyclin binding and activating phosphorylation on Thr-169 by Cak1p. Experiments using a variety of methods to resolve Thr-169 phosphorylated from unphosphorylated Cdc28p have concluded that essentially all of the Cdc28p in the cell is phosphorylated on Thr-169 throughout the cell cycle.^87,97,132 Cdc28p is found in three forms in the cell:

1. as a monomer [inactive],
2. as a heterodimer with cyclins [active], and
3. as a heterotrimer with cyclins and inhibitors [inactive].

Other potential forms would be a heterodimer with inhibitors [inactive] and a high molecular form in complex with chaperones like Cdc37p [inactive]. Interestingly, there is a 10-fold excess of Cdc28p over cyclin molecules in any given phase of the cell cycle. The monomeric pool of Cdc28p, which comprises 80–90% of the total, is as highly phosphorylated as the pool of active Cdc28p/cyclin complexes,^97,133 a surprising observation because monomeric CDKs in higher eukaryotes are not phosphorylated.^16,134 Cak1p phosphorylates monomeric CDKs much more efficiently than CDK/cyclin complexes, and both Clb and Cln cyclins associate more strongly with prephosphorylated Cdc28p than with the unphosphorylated form.^77,97 Taken together, these results suggest that the pool of phosphorylated monomers seen in vivo is likely to represent Cak1p phosphorylated monomers, and that Cdc28p is activated by subsequent binding of cyclin to phosphorylated Cdc28p. Under the conditions investigated, dephosphorylation of Thr-169 has not been shown to be a significant mechanism of Cdc28p regulation.^82,97,124 The Thr-169 phosphatases, Ptc2p and Ptc3p, act primarily on monomeric Cdc28p¹²⁴ and since there is a large pool of phosphorylated Cdc28p monomers, a limiting effect can be observed only when Cak1p function is compromised [see also the section on Removal of the Activating Phosphorylation from Cdc28p].¹²⁴

Studies on the interplay of Cdc28p regulatory mechanisms indicate that Cdc28p monomers are phosphorylated on Thr-169 by Cak1p soon after their synthesis. As cyclins accumulate, they bind to and activate the phosphorylated monomers. Cdc28p activity is switched off either through the destruction of the cyclin subunit or by binding of inhibitors to the active complex. Cdc28p activity can be further modulated through inhibitory phosphorylation, probably of the Thr-169 phosphorylated, cyclin-bound form, in response to the budding checkpoint signal, or through dephosphorylation of Cdc28p monomers by the Thr-169 phosphatases.

Although the fundamental mechanisms of Cdc28p regulation are conserved in other eukaryotes, certain aspects of the CDK activation pathway appear not to be. CDC2 and CDK2 in mammals are phosphorylated by CAK only after binding to cyclin.^{16,77,81,134,135} Budding yeast Cak1p and the CDK7/cyclin H family of CAKs that phosphorylate CDC2 and CDK2 have divergent biochemical properties that suit them to their respective activation pathways. For example, Cak1p prefers to phosphorylate monomeric CDKs, whereas CDK7/cyclin H CAKs prefer to phosphorylate CDK/cyclin complexes.^77,81 Also, Cak1p is found primarily in the cytoplasm,⁹⁵ colocalizing with the large pool of monomeric Cdc28p,⁹⁸ whereas CDK7/cyclin H is found in the nucleus, where it can phosphorylate CDKs which are directed to the nucleus upon cyclin binding.^4,136–138

One possible explanation for the opposite order of activation and for the evolution of divergent CAKs, at least for the mitosis-promoting factor [MPF], CDC2, could stem from the relative importance of inhibitory phosphorylation in CDK regulation in budding yeast versus other eukaryotes. While inhibitory phosphorylation of Cdc28p is dispensable during normal cell cycles, it is critically important for the correct timing of mitosis in other organisms (for reviews, see 7, 139). Removal of the inhibitory phosphorylations from CDC2 is the final step necessary for mitotic entry in Xenopus.¹⁵ Because inhibitory phosphorylation cannot occur until after cyclin binding,¹⁵ one way to ensure that CDC2 is not prematurely activated is for activating phosphorylation to also depend on prior cyclin binding and, ideally, on prior phosphorylation of the inhibitory sites.

A second possible explanation for the unusual Cdc28p activation pathway may be differences in the behavior of the nuclear membrane during mitosis. In mammalian cells, CDKs are known to shuttle in and out of the nucleus, and nuclear envelope breakdown occurs at the beginning of mitosis (reviewed in 4, 140, 141). Budding yeast, like other fungi, carry out a closed mitosis in which the nuclear envelope does not break down. However, CAK-phosphorylated CDK/cyclin complexes are found both in the nucleus and the cytoplasm. To achieve this, without mixing their cytoplasmic and nuclear contents, budding yeast must either have CAK activity in both compartments or, as appears to be the case, phosphorylate their CDKs as monomers in the cytoplasm before they enter the nucleus. Intriguingly, S. pombe, which also has a closed mitosis, appears to have two CAKsa CDK7/cyclin H-type CAKs (Mcs6/Mcs2) and a Cak1p-type CAK (Csk1), although the physiological roles of these two enzymes have not yet been resolved.^73,75,76,142

Future experiments aimed at understanding why different CDK activation pathways developed and continue to persist will give us insight into the complex and subtle variety of strategies of growth regulation used by eukaryotes. Results from such studies might explain the difference between CDK7-like and Cak1p-like CAKs.

Acknowledgments

We thank Mark Solomon for support, comments on the manuscript, and communication of unpublished results. Mike Shaber and Jonathan Kimmelman are acknowledged for communicating unpublished results.

P.K. thanks Karen Vousden and the NCI for support. A.C. is a Leukemia and Lymphoma Society fellow.

References

1.

King RW, Deshaies RJ, Peters J -M, Kirschner MW. How proteolysis drives the cell cycle. Science. 1996;274:1652–1659. [PubMed: 8939846]

2.

Morgan DO. The dynamics of cyclin dependent kinase structure. Curr Opin Cell Biol. 1996;8:767–772. [PubMed: 8939669]

3.

Morgan DO. Cyclin-dependent kinases: Engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261–291. [PubMed: 9442875]

4.

Pines J. Cyclins and cyclin-dependent kinases: A biochemical view. Biochem J. 1995;308:697–711. [PMC free article: PMC1136782] [PubMed: 8948422]

5.

Sherr CJ, Roberts JM. Inhibitors of mammalian G₁ cyclin-dependent kinases. Genes Dev. 1995;9:1149–1163. [PubMed: 7758941]

6.

Sherr CJ, Roberts JM. CDK inhibitors: Positive and negative regulators of G₁-phase progression. Genes Dev. 1999;13:1501–1512. [PubMed: 10385618]

7.

Solomon MJ, Kaldis P. Regulation of CDKs by phosphorylation. Results Probl Cell Differ. 1998;22:79–109. [PubMed: 9670320]

8.

Peeper DS, Parker LL, Ewen ME, Toebes M, Hall FL, Xu M. et al. A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes. EMBO J. 1993;12:1947–1954. [PMC free article: PMC413416] [PubMed: 8491188]

9.

Schulman BA, Lindstrom DL, Harlow E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc Natl Acad Sci USA. 1998;95:10453–10458. [PMC free article: PMC27915] [PubMed: 9724724]

10.

Cross FR, Jacobson MD. Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin. Mol Cell Biol. 2000;20:4782–4790. [PMC free article: PMC85916] [PubMed: 10848604]

11.

Diehl JA, Zindy F, Sherr CJ. Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev. 1997;11:957–972. [PubMed: 9136925]

12.

Draviam VM, Orrechia S, Lowe M, Pardi R, Pines J. The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus. J Cell Biol. 2001;152:945–958. [PMC free article: PMC2198800] [PubMed: 11238451]

13.

Pines J, Hunter T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 1994;13:3772–3781. [PMC free article: PMC395290] [PubMed: 8070405]

14.

Pines J, Hunter T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-depenent nuclear transport. J Cell Biol. 1991;115:1–17. [PMC free article: PMC2289910] [PubMed: 1717476]

15.

Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW. Cyclin activation of p34^cdc2. Cell. 1990;63:1013–1024. [PubMed: 2147872]

16.

Solomon MJ, Lee T, Kirschner MW. Role of phosphorylation in p34^cdc2 activation: Identification of an activating kinase. Mol Biol Cell. 1992;3:13–27. [PMC free article: PMC275499] [PubMed: 1532335]

17.

Evans T, Rosenthal ET, Youngblom J, Distel D, Hunt T. Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell. 1983;33:389–396. [PubMed: 6134587]

18.

Sherr CJ. G1 phase progression: Cycling on cue. Cell. 1994;79:551–555. [PubMed: 7954821]

19.

Koch C, Nasmyth K. Cell cycle regulated transcription in yeast. Curr Opin Cell Biol. 1994;6:451–459. [PubMed: 7917338]

20.

Patton EE, Willems AR, Tyers M. Combinatorial control in the ubiquitin-dependent proteolysis: Don't Skp the F-box hypothesis. Trends Genet. 1998;14:236–243. [PubMed: 9635407]

21.

Townsley FM, Ruderman JV. Proteolytic ratchets that control progression through mitosis. Trends Cell Biol. 1998;8:238–244. [PubMed: 9695848]

22.

Tyers M, Tokiwa G, Futcher B. Comparison of the Saccharomyces cerevisiae G₁ cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins. EMBO J. 1993;12:1955–1968. [PMC free article: PMC413417] [PubMed: 8387915]

23.

Wittenberg C, Sugimoto K, Reed SI. G1-specific cyclins of S.cerevisiae: Cell cycle periodicity, regulation by mating pheromone, and association with the p34^CDC28 protein kinase. Cell. 1990;62:225–237. [PubMed: 2142620]

24.

Oehlen LJ, Cross FR. G1 cyclins CLN1 and CLN2 repress the mating factor response pathway at Start in the yeast cell cycle. Genes Dev. 1994;8:1058–1070. [PubMed: 7926787]

25.

Oehlen LJ, Jeoung DI, Cross FR. Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast. Mol Gen Genet. 1998;258:183–198. [PubMed: 9645424]

26.

Mendenhall MD, Hodge AE. Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1998;62:1191–1243. [PMC free article: PMC98944] [PubMed: 9841670]

27.

Chang F, Herskowitz I. Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell. 1990;63:999–1011. [PubMed: 2147873]

28.

Jeoung DI, Oehlen LJ, Cross FR. Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway. Mol Cell Biol. 1998;18:433–441. [PMC free article: PMC121512] [PubMed: 9418890]

29.

Peter M, Herskowitz I. Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1. Science. 1994;265:1228–1231. [PubMed: 8066461]

30.

Mendenhall MD. An inhibitor of p34^CDC28 protein kinase activity from Saccharomyces cerevisiae. Science. 1993;259:216–219. [PubMed: 8421781]

31.

Schwob E, Böhm T, Mendenhall MD, Nasmyth K. The B-type cyclin kinase inhibitor p40^S/C1 controls the G1 to S transition in S. cerevisiae. Cell. 1994;79:233–244. [PubMed: 7954792]

32.

Donovan JD, Toyn JH, Johnson AL, Johnston LH. P40^SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae. Genes Dev. 1994;8:1640–1653. [PubMed: 7958845]

33.

Toyn JH, Johnson AL, Donovan JD, Toone WM, Johnston LH. The Swi5 transcription factor of Saccharomyces cerevisiae has a role in exit from mitosis through induction of the cdk-inhibitor Sic1 in telophase. Genetics. 1997;145:85–96. [PMC free article: PMC1207787] [PubMed: 9017392]

34.

El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM. et al. WAF1, a potent mediator of p53 tumor suppression. Cell. 1993;75:817–825. [PubMed: 8242752]

35.

Gu Y, Turck CW, Morgan DO. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature. 1993;366:707–710. [PubMed: 8259216]

36.

Harper JW, Adami GR, Wei N, Keyomarski K, Elledge SJ. The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993;75:805–816. [PubMed: 8242751]

37.

Polyak K, Kato J -y, Solomon MJ, Sherr CJ, Massagué J, Roberts JM. et al. p27^Kip1, a cyclin-cdk inhibitor, links transforming growth factor-b and contact inhibition to cell cycle arrest. Genes & Dev. 1994;8:9–22. [PubMed: 8288131]

38.

Polyak K, Lee M -H, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P. et al. Cloning of p27^Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994;78:59–66. [PubMed: 8033212]

39.

Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell. 1994;78:67–74. [PubMed: 8033213]

40.

Lee MH, Reynisdóttir I, Massagué J. Cloning of p57^KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes & Dev. 1995;9:639–649. [PubMed: 7729683]

41.

Matsuoka S, Edwards M, Bai C, Parker S, Zhang P, Baldini A. et al. p57^KIP2, a structurally distinct member of the p21^CIP1 cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev. 1995;9:650–662. [PubMed: 7729684]

42.

Hannon GJ, Beach D. p15^INK4B is a potential effector of TGF-b-induced cell cycle arrest. Nature. 1994;371:257–261. [PubMed: 8078588]

43.

Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–707. [PubMed: 8259215]

44.

Guan K, Jenkins CW, Li Y, Nichols MA, Wu X, O'Keefe CL. et al. Growth suppression by p18, A p16^INK4/MTS1- and p14^INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function. Genes Dev. 1994;8:2939–2952. [PubMed: 8001816]

45.

Chan F K M, Zhang J, Chen L, Shapiro DN, Winoto A. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16^ink4. Mol Cell Biol. 1995;15:2682–2688. [PMC free article: PMC230498] [PubMed: 7739548]

46.

Hirai H, Roussel MF, Kato J -y, Ashmun RA, Sherr CJ. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol Cell Biol. 1995;15:2672–2681. [PMC free article: PMC230497] [PubMed: 7739547]

47.

Coleman TR, Dunphy WG. Cdc2 regulatory factors. Curr Opin Cell Biol. 1994;6:877–882. [PubMed: 7880537]

48.

Amon A, Surana U, Muroff I, Nasmyth K. Regulation of p34^CDC28 tyrosine phosphorylation is not required for entry into mitosis in S.cerevisiae. Nature. 1992;355:368–371. [PubMed: 1731251]

49.

Sorger PK, Murray AW. S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34^CDC28. Nature. 1992;355:365–368. [PubMed: 1731250]

50.

Lew DJ. Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae. Curr Opin Genet Dev. 2000;10:47–53. [PubMed: 10679396]

51.

Johnson LN, Noble M E M, Owen DJ. Active and inactive protein kinases: Structural basis for regulation. Cell. 1996;85:149–158. [PubMed: 8612268]

52.

Espinoza FH, Farrell A, Erdjument-Bromage H, Tempst P, Morgan DO. A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK. Science. 1996;273:1714–1717. [PubMed: 8781234]

53.

Fesquet D, Labbé J -C, Derancourt J, Capony J -P, Galas S, Girard F. et al. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. EMBO J. 1993;12:3111–3121. [PMC free article: PMC413577] [PubMed: 8344251]

54.

Kaldis P, Sutton A, Solomon MJ. The cdk-activating kinase (CAK) from budding yeast. Cell. 1996;86:553–564. [PubMed: 8752210]

55.

Poon R Y C, Yamashita K, Adamczewski JP, Hunt T, Shuttleworth J. The cdc2-related protein p40^MO15 is the catalytic subunit of a protein kinase that can activate p33^cdk2 and p34^cdc2. EMBO J. 1993;12:3123–3132. [PMC free article: PMC413578] [PubMed: 8393783]

56.

Solomon MJ, Harper JW, Shuttleworth J. CAK, the p34^cdc2 activating kinase, contains a protein identical or closely related to p40^MO15. EMBO J. 1993;12:3133–3142. [PMC free article: PMC413579] [PubMed: 8344252]

57.

Thuret J -Y, Valay J -G, Faye G, Mann C. Civ1 (CAK in vivo), a novel Cdk-activating kinase. Cell. 1996;86:565–576. [PubMed: 8752211]

58.

Kaldis P. The cdk-activating kinase: From yeast to mammals. Cell Mol Life Sci. 1999;55:284–296. [PubMed: 10188587]

59.

Elledge SJ, Spottswood MR. A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J. 1991;10:2653–2659. [PMC free article: PMC452966] [PubMed: 1714386]

60.

Simon M, Seraphin B, Faye G. Kin28, a yeast split gene coding for a putative protein kinase homologous to CDC28. EMBO J. 1986;5:2697–2701. [PMC free article: PMC1167171] [PubMed: 3536482]

61.

Valay JG, Simon M, Faye G. The Kin28 protein kinase is associated with a cyclin in Saccharomyces cerevisiae. J Mol Biol. 1993;234:307–310. [PubMed: 8230216]

62.

Cismowski MJ, Laff GM, Solomon MJ, Reed SI. KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity. Mol Cell Biol. 1995;15:2983–2992. [PMC free article: PMC230529] [PubMed: 7760796]

63.

Valay J -G, Simon M, Dubois M -F, Bensaude O, Facca C, Faye G. The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD. J Mol Biol. 1995;249:535–544. [PubMed: 7783209]

64.

Feaver WJ, Svejstrup JQ, Henry NL, Kornberg RD. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell. 1994;79:1103–1109. [PubMed: 8001136]

65.

Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR. et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000;403:623–627. [PubMed: 10688190]

66.

Wagner M, Pierce M, Winter E. The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae. EMBO J. 1997;16:1305–1317. [PMC free article: PMC1169728] [PubMed: 9135146]

67.

Sutton A, Freiman R. The Cak1p protein kinase is required at G₁/S and G₂/M in the budding yeast cell cycle. Genetics. 1997;147:57–71. [PMC free article: PMC1208122] [PubMed: 9286668]

68.

Valay JG. Etude génétique et physiologique de la protéine kinase Kin28p de Saccharomyces cerevisiaeParis: University of Paris VI, 1994.

69.

Chun KT, Goebl MG. Mutational analysis of Cak1p, an essential protein kinase that regulates cell cycle progression. Mol Gen Genet. 1997;256:365–375. [PubMed: 9393434]

70.

Fernandez-Sarabia MJ, Sutton A, Zhong T, Arndt KT. SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2 and HCS26 RNAs during late G1. Genes & Dev. 1992;6:2417–2428. [PubMed: 1334024]

71.

Sutton A, Immanuel D, Arndt KT. The Sit4 protein phosphatase functions in late G1 for progression into S phase. Mol Cell Biol. 1991;11:2133–2148. [PMC free article: PMC359901] [PubMed: 1848673]

72.

Faye G, Valay JG, Mann C, Thuret J -Y. patent application WO 99/07836, PCT/FR 98/01788. 1999. [PMC free article: PMC140126]

73.

Hermand D, Pihlak A, Westerling T, Damagnez V, Vanderhaute J, Cottarel G. et al. Fission yeast Csk1 is a CAK-activating kinase (CAKAK). EMBO J. 1998;17:7230–7238. [PMC free article: PMC1171069] [PubMed: 9857180]

74.

Umeda M, Bhalerao RP, Schell J, Uchimiya H, Koncz C. A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana. Proc Natl Acad Sci USA. 1998;95:5021–5026. [PMC free article: PMC20206] [PubMed: 9560221]

75.

Lee KM, Saiz JE, Barton WA, Fisher RP. Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs). Curr Biol. 1999;9:441–444. [PubMed: 10226032]

76.

Hermand D, Westerling T, Pihlak A, Thuret J -Y, Vallenius T, Tiainen M. et al. Specificity of cdk activation in vivo by the two CAKs Mcs6 and Csk1 in fission yeast. EMBO J. 2001;20:82–90. [PMC free article: PMC140202] [PubMed: 11226158]

77.

Kaldis P, Russo AA, Chou HS, Pavletich NP, Solomon MJ. Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. Mol Biol Cell. 1998;9:2545–2560. [PMC free article: PMC25525] [PubMed: 9725911]

78.

Egan EA, Solomon MJ. Cyclin-stimulated binding of Cks proteins to cyclin-dependent kinases. Mol Cell Biol. 1998;18:3659–3667. [PMC free article: PMC108948] [PubMed: 9632748]

79.

Kaldis P, Cheng A, Solomon MJ. The effects of changing the site of activating phosphorylation in cdk2 from threonine to serine. J Biol Chem. 2000;275:32578–32584. [PubMed: 10931829]

80.

Hagopian JC, Kirtley MP, Stevenson LM, Gergis RM, Russo AA, Pavletich NP. et al. Kinetic basis for activation of cdk2/cyclin A by phosphorylation. J Biol Chem. 2001;276:275–280. [PubMed: 11029468]

81.

Fisher RP, Morgan DO. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell. 1994;78:713–724. [PubMed: 8069918]

82.

Cross FR, Levine K. Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase. Mol Cell Biol. 1998;18:2923–2931. [PMC free article: PMC110671] [PubMed: 9566911]

83.

Adamczewski JP, Rossignol M, Tassan J -P, Nigg EA, Moncollin V, Egly J -M. MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH. EMBO J. 1996;15:1877–1884. [PMC free article: PMC450106] [PubMed: 8617234]

84.

Roy R, Adamczewski JP, Seroz T, Vermeulen W, Tassan J -P, Schaeffer L. et al. The MO15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor. Cell. 1994;79:1093–1101. [PubMed: 8001135]

85.

Serizawa H, Mäkelä TP, Conaway JW, Conaway RC, Weinberg RA, Young RA. Association of Cdk-activating kinase subunits with transcription factor TFIIH. Nature. 1995;374:280–282. [PubMed: 7885450]

86.

Shiekhattar R, Mermelstein F, Fisher RP, Drapkin R, Dynlacht B, Wessling HC. et al. Cdk-activating kinase complex is a component of human transcription factor TFIIH. Nature. 1995;374:283–287. [PubMed: 7533895]

87.

Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO. Cak1 is required for Kin28 phosphorylation and activation in vivo. Mol Cell Biol. 1998;18:6365–6373. [PMC free article: PMC109222] [PubMed: 9774652]

88.

Kimmelman J, Kaldis P, Hengartner CJ, Laff GM, Koh SS, Young RA. et al. Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p. Mol Cell Biol. 1999;19:4774–4787. [PMC free article: PMC84276] [PubMed: 10373527]

89.

Faye G, Simon M, Valay JG, Fesquet D, Facca C. Rig2, a RING finger protein that interacts with the Kin28/Ccl1 CTD kinase in yeast. Mol Gen Genet. 1997;255:460–466. [PubMed: 9294030]

90.

Feaver WJ, Henry NL, Wang Z, Wu X, Svejstrup JQ, Bushnell DA. et al. Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. J Biol Chem. 1997;272:19319–19327. [PubMed: 9235928]

91.

Hemmer W, McGlone M, Tsigelny I, Taylor SS. Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase. J Biol Chem. 1997;272:16946–16954. [PubMed: 9202006]

92.

Enke DA, Kaldis P, Holmes JK, Solomon MJ. The cdk-activating kinase (Cak1p) from budding yeast has an unusual ATP-binding pocket. J Biol Chem. 1999;274:1949–1956. [PubMed: 9890950]

93.

Gibbs CS, Zoller MJ. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interaction. J Biol Chem. 1991;266:8923–8931. [PubMed: 2026604]

94.

Enke DA, Kaldis P, Solomon MJ. Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast. J Biol Chem. 2000;275:33267–33271. [PubMed: 10934199]

95.

Kaldis P, Pitluk ZW, Bany IA, Enke DA, Wagner M, Winter E. et al. Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast. J Cell Sci. 1998;111:3585–3596. [PubMed: 9819350]

96.

Russo AA, Jeffrey PD, Patten AK, Massagué J, Pavletich NP. Crystal structure of the p27^Kip1 cyclin-dependent kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature. 1996;382:325–331. [PubMed: 8684460]

97.

Ross KE, Kaldis P, Solomon MJ. Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, Cdc28p, precedes cyclin binding. Mol Biol Cell. 2000;11:1597–1609. [PMC free article: PMC14870] [PubMed: 10793138]

98.

Wittenberg C, Richardson SL, Reed SI. Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: Evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix. J Cell Biol. 1987;105:1527–1538. [PMC free article: PMC2114673] [PubMed: 3312233]

99.

Kupiec M, Byers B, Esposito R, Mitchell A. Meiosis and sporulation in Saccharomyces cerevisiaeIn: Pringle J, Broach J, Jones E, eds. The Molecular and Cellular Biology of the Yeast SaccharomycesCold Spring Harbor: Cold Spring Harbor Press,19978891036.

100.

Shuster EO, Byers B. Pachytene arrest and other meiotic effects of the start mutations in Saccharomyces cerevisiae. Genetics. 1989;123:29–43. [PMC free article: PMC1203788] [PubMed: 2680756]

101.

Dirick L, Goetsch L, Ammerer G, Byers B. Regulation of meiotic S phase by Ime2 and a Clb5,6-associated kinase in Saccharomyces cerevisiae. Science. 1998;281:1854–1857. [PubMed: 9743499]

102.

Stuart D, Wittenberg C. CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint. Genes Dev. 1998;12:2698–2710. [PMC free article: PMC317137] [PubMed: 9732268]

103.

Dirick L, Böhm T, Nasmyth K. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J. 1995;14:4803–4813. [PMC free article: PMC394578] [PubMed: 7588610]

104.

Schneider BL, Yang Q -H, Futcher AB. Linkage of replication to start by the cdk inhibitor Sic1. Science. 1996;272:560–562. [PubMed: 8614808]

105.

Tyers M. The cyclin-dependent kinase inhibitor p40^SIC1 imposes the requirement for Cln G1 cyclin function at Start. Proc Natl Acad Sci USA. 1996;93:7772–7776. [PMC free article: PMC38823] [PubMed: 8755551]

106.

Verma R, Annan RS, Huddleston MJ, Carr SA, Reynard G, Deshaies RJ. Phosphorylation of Sic1p by G₁ cdk required for its degradation and entry into S phase. Science. 1997;278:455–460. [PubMed: 9334303]

107.

Dahmann C, Futcher B. Specialization of B-type cyclins for mitosis or meiosis in S.cerevisiae. Genetics. 1995;140:957–963. [PMC free article: PMC1206679] [PubMed: 7672594]

108.

Roeder GS, Bailis JM. The pachytene checkpoint. Trends Genet. 2000;16:395–403. [PubMed: 10973068]

109.

Leu JY, Roeder GS. The pachytene checkpoint in S.cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28. Mol Cell. 1999;4:805–814. [PubMed: 10619027]

110.

Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown PO. et al. The transcriptional program of sporulation in budding yeast[published erratum appears in Science 1998 Nov 20; 282(5393):1421]. Science 1998282699–705. [PubMed: 9784122]

111.

Primig M, Williams RM, Winzeler EA, Tevzadze GG, Conway AR, Hwang SY. et al. The core meiotic transcriptome in budding yeasts. Nat Genet. 2000;26:415–423. [PubMed: 11101837]

112.

Chu S, Herskowitz I. Gametogenesis in yeast is reglated by a transcriptional cascade dependent on Ndt80. Mol Cell. 1998;1:685–696. [PubMed: 9660952]

113.

Hepworth SR, Friesen H, Segall J. NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae. Mol Cell Biol. 1998;18:5750–5761. [PMC free article: PMC109161] [PubMed: 9742092]

114.

Tung KS, Hong EJ, Roeder GS. The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80. Proc Natl Acad Sci USA. 2000;97:12187–12192. [PMC free article: PMC17316] [PubMed: 11035815]

115.

Neiman AM, Stevenson BJ, Xu HP, Sprague G F Jr, Herskowitz I, Wigler M. et al. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms. Mol Biol Cell. 1993;4:107–120. [PMC free article: PMC300904] [PubMed: 8443406]

116.

Krisak L, Strich R, Winters RS, Hall JP, Mallory MJ, Kreitzer D. et al. SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes Dev. 1994;8:2151–2161. [PubMed: 7958885]

117.

Wagner M, Briza P, Pierce M, Winter E. Distinct steps in yeast spore morphogenesis require distinct SMK1 MAP kinase thresholds. Genetics. 1999;151:1327–1340. [PMC free article: PMC1460549] [PubMed: 10101160]

118.

Gould KL, Moreno S, Owen DJ, Sazer S, Nurse P. Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34^cdc2 function. EMBO J. 1991;10:3297–3309. [PMC free article: PMC453056] [PubMed: 1655416]

119.

Lorca T, Labbé J -C, Devault A, Fesquet D, Capony J -P, Cavadore J -C. et al.Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase EMBO J 1992112381–2390. [PMC free article: PMC556712] [PubMed: 1321030]

120.

Lee TH, Solomon MJ, Mumby MC, Kirschner MW. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell. 1991;64:415–423. [PubMed: 1846321]

121.

Gyuris J, Golemis E, Chertkov H, Brent R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 1993;75:791–803. [PubMed: 8242750]

122.

Hannon GJ, Casso D, Beach D. KAP: A dual specificity phosphatase that interacts with cyclin- dependent kinases. Proc Natl Acad Sci USA. 1994;91:1731–1735. [PMC free article: PMC43237] [PubMed: 8127873]

123.

Poon Y C R, Hunter T. Dephosphorylation of Cdk2 Thr¹⁶⁰ by the cyclin-dependent kinase-interacting phosphatase KAP in the absence of cyclin. Science. 1995;270:90–93. [PubMed: 7569954]

124.

Cheng A, Ross KE, Kaldis P, Solomon MJ. Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases. Genes & Dev. 1999;13:2946–2957. [PMC free article: PMC317162] [PubMed: 10580002]

125.

Cheng A, Kaldis P, Solomon MJ. Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2Ca and b2 isoforms. J Biol Chem. 2000;275:34744–34749. [PubMed: 10934208]

126.

Kobayashi T, Kusuda K, Ohnishi M, Wang H, Ikeda S, Hanada M. et al. Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells. FEBS Lett. 1998;430:222–226. [PubMed: 9688543]

127.

Nishikawa M, Omay SB, Nakai K, Kihira H, Kobayashi T, Tamura S. et al. Up-regulation of protein serine/threonine phosphatase type 2C during 1 alpha,25-dihydroxyvitamin D3-induced monocytic differentiation of leukemic HL-60 cells. FEBS Lett. 1995;375:299–303. [PubMed: 7498522]

128.

Barral Y, Parra M, Bidlingmaier S, Snyder M. Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. Genes Dev. 1999;13:176–187. [PMC free article: PMC316392] [PubMed: 9925642]

129.

Lew DJ, Reed SI. A cell cycle checkpoint monitors cell morphology in budding yeast. J Cell Biol. 1995;129:739–749. [PMC free article: PMC2120431] [PubMed: 7730408]

130.

Sia RA, Herald HA, Lew DJ. Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast. Mol Biol Cell. 1996;7:1657–1666. [PMC free article: PMC276016] [PubMed: 8930890]

131.

Peter M, Gartner A, Horecka J, Ammererer G, Herskowitz I. FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell. 1993;73:747–760. [PubMed: 8500168]

132.

Hadwiger JA, Reed SI. Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase. Mol Cell Biol. 1988;8:2976–2979. [PMC free article: PMC363517] [PubMed: 3043202]

133.

Wittenberg C, Reed SI. Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex. Cell. 1988;54:1061–1072. [PubMed: 3046752]

134.

Desai D, Gu Y, Morgan DO. Activation of human cyclin-dependent kinases in vitro. Mol Biol Cell. 1992;3:571–582. [PMC free article: PMC275609] [PubMed: 1535244]

135.

Desai D, Wessling HC, Fisher RP, Morgan DO. The effect of phosphorylation by CAK on cyclin binding by CDC2 and CDK2. Mol Cell Biol. 1995;15:345–350. [PMC free article: PMC231966] [PubMed: 7799941]

136.

Jordan P, Cunha C, Carmo-Fonseca M. The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies. Mol Biol Cell. 1997;8:1207–1217. [PMC free article: PMC276147] [PubMed: 9243502]

137.

Tassan J -P, Schultz SJ, Bartek J, Nigg EA. Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). J Cell Biol. 1994;127:467–478. [PMC free article: PMC2120215] [PubMed: 7929589]

138.

Tassan J -P, Jaquenoud M, Fry AM, Frutiger S, Hughes GJ, Nigg EA. In vitro assembly of a functional human CDK7-cyclin H complex requires MAT1, a novel 36 kDa RING finger protein. EMBO J. 1995;14:5608–5617. [PMC free article: PMC394676] [PubMed: 8521818]

139.

Lew DJ, Kornbluth S. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 1996;8:795–804. [PubMed: 8939679]

140.

Pines J. Four-dimensional control of the cell cycle. Nat Cell Biol. 1999;1:E73–E79. [PubMed: 10559915]

141.

Yang J, Kornbluth S. All aboard the cyclin train: Subcellular trafficking of cyclins and their cdk partners. Trends Cell Biol. 1999;9:207–210. [PubMed: 10354564]

142.

Damagnez V, Mäkelä TP, Cottarel G. Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. EMBO J. 1995;14:6164–6172. [PMC free article: PMC394741] [PubMed: 8557036]