Considerable genetic and biochemical evidence indicates that Cdc28 is phosphorylated and activated by Cak1. Defects in CAK1
function, as in our cak1
mutants or in the previously described cak1-22
allele, enhance the growth defects of cdc28
mutants (Table ) and cells deficient in various Cdc28 cyclins (22
). Cdc28 phosphorylation and activity are greatly reduced in conditional cak1
) (Fig. A), and the Cak1 protein is able to catalyze Cdc28 phosphorylation in vitro (10
). Thus, it appears likely that Cak1 is directly responsible for the activating phosphorylation of Cdc28 in the cell.
The present work suggests that Cak1 is also required for the phosphorylation and activity of Kin28. Mutations in CAK1
greatly exacerbate the growth defect observed in the kin28-3
mutant (Table ) (54
). Kin28 phosphorylation in vivo is decreased in conditional cak1
mutants and in cells lacking the CAK1
gene, and coexpression of Cak1 and Kin28 in insect cells results in Kin28 phosphorylation and activation. These data are most consistent with a direct role for Cak1 in the phosphorylation of Kin28 in vivo, although clear evidence for direct phosphorylation will require reconstitution of Kin28 phosphorylation by purified Cak1 in vitro.
In addition to causing defects in Kin28 phosphorylation, mutations in CAK1
caused decreased Kin28 protein levels. These decreases occurred gradually at the restrictive temperature (over a period of 3 h [data not shown]). Considering the requirement for KIN28
in mRNA synthesis (4
), we suspect that decreases in Kin28 protein levels reflect a decrease in KIN28
transcription that is secondary to the defect in Kin28 activation by phosphorylation. Interestingly, the decrease in Kin28 protein levels in cak1-23
cells at 37°C was not accompanied by major changes in Kin28 phosphorylation, suggesting that Kin28 phosphorylation was relatively stable under these conditions.
The arrest phenotypes of cak1
mutants can be roughly divided into two classes. Our cak1-95
mutant, like the previously described cak1-4
) mutant, arrests primarily as unbudded G1
). On the other hand, our cak1-23
mutants, as well as the previously described cak1-22
mutant and cak1
Δ cells (3
), arrest with a mixture of phenotypes that includes unbudded cells and cells with elongated buds. The two classes of cak1
arrest phenotypes are not simply the result of differences in the bulk kinase activity of Cdc28 or Kin28; for example, cak1-34
mutants contain similar levels of these kinase activities but arrest at 37°C with different phenotypes. Instead, variations in cak1
phenotypes may be due in part to differences in the ability of mutant Cak1 proteins to target different CDK-cyclin complexes. For example, the elongated budding phenotype of cak1-23
resembles that seen in cells with compromised Clb-Cdc28 kinase activity (1
) while the unbudded phenotype of cak1-95
is reminiscent of the G1
arrest seen in cdc28
mutants or in cells lacking the G1
cyclins Cln1 to Cln3 (6
). The complexity of cak1
phenotypes may also reflect differences in the stability of Cak1-dependent CDK activities: we found that shifting cak1
mutant cells to 37°C caused a more rapid loss of Cdc28 activity (10 min) than of Kin28 activity (1 to 2 h). Thus, the major features of the cak1
phenotype may be due in large part to defects in specific Cdc28-cyclin functions but the gradual loss of KIN28
-dependent transcriptional activity in these mutants may complicate the phenotype.
Cells carrying Cak1-independent Cdc28 mutants and lacking CAK1 are able to proliferate (at reduced rates) even though the Kin28-associated CTD kinase activity is greatly reduced, indicating that remarkably little Kin28 activity is required for cell viability (assuming that the essential function of Kin28 is dependent on its kinase activity). Furthermore, the absence of detectable Kin28 phosphorylation in these cells indicates that the essential function of Kin28 does not require its phosphorylation. This result seems to contradict our evidence that overexpression of Kin28A does not allow growth of kin28-3 cells at high temperature (Fig. C). Perhaps Kin28 in the Cak1-independent cells is phosphorylated by some other kinase at a very low level that is not detectable by our methods but is still sufficient to provide the small amount of Kin28 activity required for cell proliferation. Alternatively, the Kin28A mutant may be defective not only in activating phosphorylation but also in another essential biochemical function.
Our studies with the Kin28A mutant, as well as those with cak1
-deficient cells, clearly suggest that phosphorylation of Kin28 at T162 is required for full kinase activity in vivo. Thus, the Kin28-Ccl1 complex appears to be more dependent on phosphorylation than is its vertebrate homologue, Cdk7-cyclin H, whose activation does not require phosphorylation in the presence of the assembly factor Mat1 (9
). The S. cerevisiae
homologue of Mat1, Tfb3, may not provide the same activating function as Mat1 in the absence of phosphorylation. Indeed, in our insect cell coinfection experiments, we found that coexpression of Kin28, Ccl1, and Tfb3 did not yield an active kinase complex except in the presence of Cak1, suggesting that activating phosphorylation is required even in the presence of Tfb3. Nevertheless, Tfb3 and Ccl1 were required for maximal Kin28 phosphorylation and activity in these experiments. Thus, Cak1 does not appear to promote the phosphorylation of monomeric Kin28, despite its ability to phosphorylate the Cdc28 monomer (10
). Further studies, preferably with purified components in vitro, are required to assess the precise role of Tfb3 and Ccl1 in Kin28 assembly, phosphorylation, and substrate targeting.
Our strongest cak1
) did not affect the kinase activity, mobility, or levels of Pho85 or Srb10. Similarly, several mutations in CAK1
do not affect the expression of PHO5
, a gene whose expression increases in the absence of PHO85
function (reference 47
and data not shown). These results raise the possibility that Pho85 and Srb10 are activated by a different CAK or that their activation does not require phosphorylation. There is little previous data to shed light on these possibilities. For Pho85, mutation of the putative activating site (Ser166) reduces kinase activity and function in vivo, but phosphorylation at this site has not been demonstrated (41
). Similarly, nothing is known about the phosphorylation of Srb10; interestingly, its putative human homologue, Cdk8, does not contain a phosphorylatable residue in the T-loop region (29
). It is therefore possible that phosphorylation is not necessary for the activation of Pho85 or Srb10. Perhaps nonessential CDKs like these can evolve more easily through the intermediate steps between phosphorylation dependence and independence; these steps may be insurmountable in essential CDKs like Kin28 and Cdc28 (5
Cak1 may also be involved in the activation of kinases other than CDKs. Overexpression of CAK1
suppresses the spore wall defect of cells with mutations in SMK1
, a gene encoding a member of the mitogen-activated protein kinase family (24
). Cells with defects in CAK1
exhibit a spore wall formation defect that resembles the phenotype of smk1
). Like the closely related CDKs, mitogen-activated protein kinases are activated by phosphorylation of residues within the activating loop. Smk1 contains a threonine (T207) in a position that is roughly analogous to the activating site of CDKs (24
). Thus, the requirement for Cak1 in spore wall formation may reflect a direct role in the activation of Smk1.
In vertebrates and other higher eukaryotes, the Cdk7-cyclin H-Mat1 complex comprises the major CAK activity in cell lysates, and recent evidence suggests that Drosophila melanogaster cdk7
mutants are defective in the activation of the mitosis-promoting kinase Cdc2 (28
). Because of its association with TFIIH, Cdk7 is also thought to contribute to the control of CTD phosphorylation and transcription. Thus, Cdk7 appears to fulfill dual roles in CDK activation and transcription in higher eukaryotes, while its budding yeast homologue Kin28 is involved primarily in transcriptional control. Our results now demonstrate that yeast Cak1 also plays two roles, both in the activation of cell cycle progression through Cdc28 and in the activation of transcription through Kin28 (Fig. ). This scheme raises the possibility that a higher eukaryotic homologue of Cak1 is responsible for the phosphorylation of Cdk7.
Regulatory pathways governing the activities of Kin28 and Cdc28 in S. cerevisiae (left), and homologous pathways in higher eukaryotes (right).