Evolution of Cyclin-Dependent Kinases (CDKs) and CDK-Activating Kinases (CAKs): Differential Conservation of CAKs in Yeast and Metazoa

Ji Liu and Edward T. Kipreos2,

Department of Cellular Biology, University of Georgia


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs. Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date. To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans. We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes. The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans. In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85. One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence. Interestingly, CAK1 and csk1 branch together with high bootstrap support values. We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction. This result suggests that CAK1 and csk1 are orthologs and that a central aspect of CAK regulation has been conserved in budding and fission yeast. Although there are metazoan CDK-family members for which we could not define ancestral lineage, our analysis failed to identify metazoan CAK1/csk1 orthologs, suggesting that if the CAK1/csk1 gene existed in the metazoan ancestor, it has not been conserved.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Cyclin-dependent kinases (CDKs) are serine/threonine kinases that must bind to cyclin proteins to become active (Pines 1995Citation ). They were originally identified as essential regulators of cell cycle progression. CDKs are required for the G1-to-S phase cell cycle transition, initiation of DNA replication, the G2-to-M phase cell cycle transition, and initiation of multiple mitotic events (King, Jackson, and Kirschner 1994Citation ; Sherr 1994Citation ; Stillman 1996Citation ). The first CDKs to be identified were the budding yeast cell cycle regulator CDC28 and the orthologous fission yeast cell cycle regulator cdc2 (Nasmyth and Reed 1980Citation ; Beach, Durkacz, and Nurse 1982Citation ; Hindley and Phear 1984Citation ; Lorincz and Reed 1984Citation ). There is an extended eukaryotic family of CDKs that share homology with CDC28 and cdc2. While certain CDK family members function to regulate the cell cycle, other CDKs have been found to function in other cellular pathways, most notably as central regulators of transcription (Morgan 1997Citation ). The functions of many CDKs are still unknown (Morgan 1997Citation ).

In both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, a single CDK (Cdc28 and Cdc2, respectively), is responsible for catalyzing all major cell cycle transitions (Morgan 1997Citation ). In higher eukaryotes, there has been an expansion in the number of CDKs that regulate the cell cycle, with five cell cycle CDKs in mammals. This expansion allowed the specialization of CDKs for particular cell cycle transitions/functions: CDK4, CDK6, and CDK3 regulate G1 phase progression and entry into S phase; CDK2 is required for entry into S phase and DNA replication; and CDK1 (CDC2) is required for mitosis (van den Heuvel and Harlow 1993Citation ; King, Jackson, and Kirschner 1994Citation ; Sherr 1994Citation ; Stillman 1996Citation ; Morgan 1997Citation ).

In S. cerevisiae, five CDKs function to regulate transcription. Three of these CDKs, Kin28, Srb10, and Ctk1, regulate mRNA synthesis by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II (Valay et al. 1995Citation ; Liao et al. 1995Citation ; Lee and Greenleaf 1991Citation ; Sterner et al. 1995Citation ). Sgv1 regulates transcription, potentially also as a CTD kinase, as its ortholog CDK9 functions as a CTD kinase (Prelich and Winston 1993Citation ; Reines, Conaway, and Conaway 1999Citation ). Finally, Pho85 functions to inhibit gene transcription in response to phosphate (Lenburg and O'Shea 1996Citation ). Pho85 also has a secondary role in promoting cell cycle progression, as it is required for G1-to-S phase progression when the G1 cyclins Cln1 and Cln2 are missing (Espinoza et al. 1994Citation ; Measday et al. 1994Citation ).

CDK activity is tightly regulated through four mechanisms: (1) binding by activating cyclins, (2) binding by inhibitory cyclin-dependent kinase inhibitors (CKIs), (3) inhibitory phosphorylation of the CDK, and (4) activating phosphorylation of the CDK. The activating phosphorylation is catalyzed by a CDK-activating kinase (CAK) (Kaldis 1999Citation ). CAK phosphorylates a conserved threonine or serine residue in CDKs (throughout this paper, this site will be referred to as Thr 160, which corresponds to the position in CDK2, although the exact amino acid position varies among the kinases). This phosphorylation stabilizes cyclin-CDK interaction and enhances substrate binding (Russo, Jeffrey, and Pavletich 1996Citation ). CDKs depend on CAK phosphorylation for activation, and a loss of CAK activity causes cell cycle arrest (Espinoza et al. 1996Citation ; Kaldis, Sutton, and Solomon 1996Citation ; Thuret et al. 1996Citation ; Larochelle et al. 1998Citation ; Lee et al. 1999Citation ).

In S. cerevisiae, Cak1/Civ1 is the sole essential CAK for both the cell cycle CDK Cdc28 and the transcriptional CDK Kin28 (Kaldis, Sutton, and Solomon 1996Citation ; Thuret et al. 1996Citation ; Espinoza et al. 1998Citation ). It is a distant member of the CDK family and has diverged considerably not only from other CDKs, but also from other kinases. Cak1 is fully active without a cyclin partner or activating phosphorylation (Kaldis, Sutton, and Solomon 1996Citation ; Thuret et al. 1996Citation ). Furthermore, it lacks the consensus GXGXXG motif that is implicated in nucleotide binding for all classes of protein kinase (Hanks and Hunter 1995Citation ). Currently, only two other kinases have been found that also lack the entire GXGXXG motif, Mik1 and Vps15 (Kaldis 1999Citation ). No CAK1 ortholog has been reported in any other organism to date.

The budding yeast CDK Kin28 possesses no CAK activity in vitro or in vivo (Feaver et al. 1994Citation ; Cismowski et al. 1995Citation ; Valay et al. 1995Citation ). However, in metazoans, the Kin28 ortholog CDK7/p40MO15 was identified as a CAK based on its ability to phosphorylate and activate CDK1 in vitro (Poon et al. 1993Citation ; Solomon, Harper, and Shuttleworth 1993Citation ; Darbon et al. 1994Citation ; Tassan et al. 1994Citation ). In Drosophila melanogaster, a Cdk7 temperature-sensitive mutant was found to be defective for CAK activity for the cdc2/Cyclin A and cdc2/Cyclin B complexes (Larochelle et al. 1998Citation ). The conclusion that Cdk7 was the in vivo CAK for cdc2 was tempered by the fact that Cdk7 also functions as a transcriptional activator, and therefore the loss of CAK activity could have resulted from an inability to synthesize the true CAK. The D. melanogaster Cdk7 mutations did not affect the activating phosphorylation of cdc2c (Cdk2) in vivo, suggesting that there are additional CAKs in metazoans (Larochelle et al. 1998Citation ). CDK7 binds Cyclin H and requires activating phosphorylation (Kaldis 1999Citation ). The in vivo CAK for CDK7 is currently not known.

In S. pombe, the Kin28 ortholog Mcs6/Mop1/Crk1 is a CAK for the cell cycle CDK, Cdc2 (Buck, Russell, and Millar 1995Citation ; Damagnez, Makela, and Cottarel 1995Citation ). A divergent CDK family member, Csk1, works redundantly with Mcs6 to activate Cdc2 (Lee et al. 1999Citation ). Csk1 also phosphorylates Mcs6 on the consensus activating site Ser 165 (Molz and Beach 1993Citation ; Hermand et al. 1998Citation ).

Cell cycle regulators are largely conserved among eukaryotes (Pines 1995Citation ). As Cak1 is an essential cell cycle regulator in budding yeast, it is perplexing that no CAK1 ortholog has been found in any other organisms. Similarly, no ortholog of fission yeast csk1 has been reported. Since the metazoan CAK CDK7 does not appear to be sufficient for the activation of all CDKs (Larochelle et al. 1998Citation ), and CDK7 itself needs activating phosphorylation, it is likely that there exists an unidentified metazoan CAK(s).

The genome sequence of Caenorhabditis elegans is essentially complete (C. elegans Sequencing Consortium 1998Citation ), allowing a comprehensive analysis of a metazoan genome. Further, the genome of D. melanogaster has also been sequenced (Adams et. al. 2000). We identified 13 D. melanogaster and 14 C. elegans extended CDK family members. We undertook a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, and metazoans to provide insight into the evolution of the CDK family and to address whether an ortholog of CAK1 could be identified in metazoans.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Identification and Alignment of Protein Sequences
Yeast, human, D. melanogaster, and C. elegans CDK protein sequences were obtained from the National Center for Biotechnology Information (NCBI), the Institute for Genomic Research (TIGR), and C. elegans genome databases. CDKs were identified with BLAST (Altschul et al. 1997Citation ) and PROFILE (Gribskov, Luthy, and Eisenberg 1990Citation ) searches of the databases. The exon structures of three D. melanogaster sequences that are not represented in the EST databases, AC017581, AC014407, and AC018104, were predicted from the genomic sequence based on kinase domain homology and splice site consensus. The predicted exons for the AC017581 kinase domain are at nucleotide positions 96285–96040, 95976–95588, and 95296–94819; the predicted exons for AC014407 are at positions 22352–22122, 22098–21714, 21651–21501, and 21448–21277; and the predicted exons for AC018104 are at positions 100085–99918 and 99860–98946. Table 1 shows the sequences used in this study.


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Table 1 Yeast, Human, and Caenorhabditis elegans CDK-Related Kinases Used in this Study

 
Initial protein sequence alignments were made with the CLUSTAL X program (Thompson, Higgins, and Gibson 1994Citation ). The alignment was then optimized by hand to minimize insertions/deletions using the kinase alignment of Hanks and Hunter (1995)Citation as a guide. Spacer regions were excluded from the phylogenetic analysis, as was the region between helix 5 and helix 6 that is not conserved among kinases (Hanks and Hunter 1995Citation ) (fig. 1 ).



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Fig. 1.—Alignment of the CDK kinase domains. Alpha helixes and beta sheets, determined from the crystal structure of human CDK2 (De Bondt et al. 1993Citation ), are presented above the sequence. Brackets over the sequence denote residues included in the phylogenetic analyses of figure 2 . Plus symbols below the sequence indicate residues in the slow-evolving character groups S3–S7, which were used in figure 3 . Zeros below the sequence indicate residues in the slowest-evolving character groups, S1 and S2. The thick bar between alpha helixes {alpha}L12 and {alpha}4 denotes the extent of the T-loop. The star denotes the position of the threonine or serine residue corresponding to Thr 160 of CDK2 that is phosphorylated by CAK. Outgroup taxa are presented separately below the CDKs

 
Based on the apparent orthology of the C. elegans CDK family members Y39G10AL, F39H11.3, and H25P06.2 with human CDKs, we assigned them the names cdk-7, cdk-8, and cdk-9, respectively. We refrained from naming C. elegans or D. melanogaster genes that were not orthologous to known cyclin-binding proteins, which are given the permanent designation CDK as per convention (Myerson et al. 1992Citation ).

Phylogenetic Analysis
The neighbor-joining (NJ) (Saitou and Nei 1987Citation ) and maximum-likelihood (ML) methods were used to create phylogenies. For NJ analyses, a distance matrix of the data set, using the JTT model, was created with the ProtML program of MOLPHY, version 2.3 (Adachi and Hasegawa 1996Citation ). The matrix was used to create trees with the NEIGHBOR program of the PHYLIP package (Felsenstein 1993Citation ), using random order of addition of taxa. Bootstrap values were obtained from 1,000 replicates.

Another JTT distance matrix was calculated using a model of rate heterogeneity among sites with eight rates following a gamma distribution plus one invariable rate using the PUZZLE program (Strimmer and von Haeseler 1996Citation ) with exact parameter estimates of the gamma distribution parameter alpha and the fraction of invariable sites. The distance matrix was analyzed with the NEIGHBOR program (Felsenstein 1993Citation ). Bootstrap values were obtained from 100 replicates.

An ML analysis was performed with the ProtML program of MOLPHY, version 2.3 (Adachi and Hasegawa 1996Citation ). Input trees derived from Star Decomposition searches (ProtML program) or NJ trees were optimized by local branch rearrangement (LBR) analysis using the JTT model (Adachi and Hasegawa 1996Citation ). We observed that LBRs of NJ input trees always gave higher likelihood phylogenies than those obtained using Star Decomposition input trees. Bootstrap probabilities were created by ProtML LBR analysis of 100 input data set resamplings created with the SeqBoot program (Felsenstein 1993Citation ) using their associated NJ trees as input.

Relative apparent synapomorphy analysis (RASA) was applied using the RASA, version 2.3, package (Lyons-Weiler, Hoelzer, and Tausch 1996Citation ; Lyons-Weiler and Hoelzer 1997Citation ; Lyons-Weiler 1999Citation ). After signal content analysis, a taxon variance analysis was used to detect taxa that manifested the hallmarks of long-branch attraction (Lyons-Weiler 1999Citation ).

The removal of fast-evolving characters was accomplished according to the S-F method as described by Brinkmann and Philippe (1999)Citation . The substitution step was calculated with PAUP, version 4.0.b2a (Swofford 1993Citation ), as described (Brinkmann and Philippe 1999Citation ).

Molecular Experiments
An H01G02.2 cDNA was isolated by PCR from a cDNA library (provided by R. Barstead) using primers designed to amplify the coding region of H01G02.2, predicted by the C. elegans Genome Consortium (the Sanger Centre, Cambridge, and the Genome Sequencing Center at the Washington University School of Medicine, St. Louis, Mo.). The H01G02.2 cDNA was cloned into the pBluescript SKII+ plasmid (Stratagene) and sequenced to confirm the absence of PCR-induced mutations. Sense and antisense H01G02.2 RNAs were synthesized using the Megascript T7 and T3 kits according to the manufacturer's instructions (Ambion). Sense and antisense RNAs were annealed to create double-stranded RNA by heating to 93°C for 30 s, followed by twenty-five 30-s incubations at 2° decrements for each incubation. H01G02.2 dsRNA was injected into young adult C. elegans hermaphrodites at a concentration of 0.5 to 1 mg/ml. F1 progeny from injected mothers were scored for abnormalities.

To test whether H01G02.2 could complement cak1/civ1 mutants, an H01G02.2 cDNA was subcloned into the GAL1 promoter yeast expression plasmid pYes2 (Invitrogen). The H01G02.2/pYes2 plasmid and the pYes2 plasmid vector control were transformed by electroporation into the cak1-22 mutant strain SY143 (cak1::HIS3 [LEU2/cen-cak1-22],ade2, his3, leu2, can1, ura3, trp1, ssd1) (Kaldis, Sutton, and Solomon 1996Citation ) and the civ1-4 mutant strain GF2351 (civ1-4, ura3, leu2, trp1, lys2, ade2, ade3) (Thuret et al. 1996Citation ). The growth of strain SY143 (cak1-22) and strain GF2351 (civ1-4) was scored at 25°C (permissive temperature) and 37°C (nonpermissive temperature) on minimal media plates supplemented with 20 mg/ml galactose (to induce the GAL1 promoter).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Using S. cerevisiae and human CDKs and CAKs as probes, BLAST and PROFILE searches were used to identify CDK family members in budding yeast, fission yeast, C. elegans, D. melanogaster, and humans. We identified 14 CDK family members for C. elegans, 13 for D. melanogaster, and 8 for S. pombe. We also included the previously identified 7 S. cerevisiae CDK family members and 21 of the known 22 human CDK family members in our analysis (human PITSLRE A, which is virtually identical to PITSLRE B, was not included). The CDK family members used in the study are listed in Table 1.

Phylogenetic Analysis
The evolutionary rates of the yeast, D. melanogaster (D.m.), C. elegans (C.e.),and human (H.s.) CDK family members were expected to be different. We therefore chose to perform our phylogenetic analysis with the ML method and the NJ distance method (Felsenstein 1981Citation ; Saitou and Nei 1987Citation ). These methods are less likely to be misled than parsimony or compatibility methods when the evolutionary rates differ among taxa (Felsenstein 1978Citation ; Saitou and Imanishi 1989Citation ). When taxa have dissimilar rates of substitutions, incorporating the differential rate of character changes at given sites by fitting the amino acid data set to gamma rate distributions facilitates a more accurate phylogeny (Yang 1996Citation ). We therefore used gamma-corrected distances for the NJ phylogeny.

The ML and gamma-corrected NJ methods both produced 10 clades for the CDKs (fig. 2 ). Seven of the clades for both methods contain both yeast and metazoan members and therefore represent ancestral clades. We will refer to these clades by their S. cerevisiae members, except for the clade containing only fission yeast BC18H10.15. Two of the clades, the CDK4/CDK6 clade and the KKIALRE clade, contain only metazoan members.



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Fig. 2.—Gamma-rate corrected neighbor-joining (NJ) phylogeny of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans, and human CDKs using the complete alignment (fig. 1 ). The amino acid data set was modeled onto one invariable and eight gamma rates to produce the pairwise distances that were used to create the NJ tree. Branch lengths are proportional to the estimated number of amino acid substitutions; the scale bar indicates amino acid substitutions per site. Bootstrap support values above 50% are given at branch nodes and are derived from maximum-likelihood (left), NJ of uncorrected data set (center), and gamma-rate-corrected NJ analyses (right), separated by slash marks. Species are denoted by cartoon. Ancestral clades are denoted by brackets on the right

 
Although both Cak1 and Csk1 function as CAKs to activate both cell cycle and transcription CDKs, it has not been recognized that CAK1 and csk1 may be orthologs (Hermand et al. 1998Citation ; Lee et al. 1999Citation ), perhaps because both genes have diverged considerably from other CDKs and from each other. All metazoan and yeast CDK family members (with the exception of CAK1 and csk1) are more similar to all other yeast CDK family members than they are to CAK1 and csk1 (table 2 ; data not shown). In contrast to the bottom ordinal ranking of similarity of other CDKs with CAK1 and csk1, CAK1 is the second most similar gene to csk1 among the seven budding yeast CDK family members, and csk1 is the fourth most similar gene to CAK1 among the eight fission yeast CDK family members (table 2 ; data not shown). Furthermore, CAK1 and csk1 branch together with significant bootstrap values (fig. 2 ).


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Table 2 JTT Distance Matrix of Caenorhabditis elegans and Budding Yeast CDK Family Members

 
A potential source of misleading convergence in trees is the problem of long-branch attraction. Long-branch attraction is the phenomenon whereby taxa that have evolved at a higher rate relative to other taxa converge in trees due to their shared dissimilarity relative to slow-evolving taxa (Felsenstein 1978Citation ). To determine if our data set included taxa that could potentially produce long-branch attraction, we employed the RASA method (Lyons-Weiler, Hoelzer, and Tausch 1996Citation ; Lyons-Weiler and Hoelzer 1997Citation ). RASA compares the cladistic similarity of taxa (RASs; relative apparent synapomorphies) with their phenetic similarity (E; overall similarity). Taxa with long-branch attraction problems have abnormally high ratios of RAS to E (Ftv's), because while the overall similarity (E) between these and other taxa is low, the fast-evolving taxa have characters that have changed to the extent that their current state is essentially random and can by chance become identical to the characters of other taxa (homoplasy), thereby generating an abnormally high RAS value. In contrast, slow-evolving taxa diverge from ancestral states slowly and are unlikely to suffer from severe homoplasy. Their shared character traits are more likely to be true synapomorphies (evolutionarily shared character traits) and will be present as a function of overall similarity. Using the RASA program (Lyons-Weiler, Hoelzer, and Tausch 1996Citation ; Lyons-Weiler and Hoelzer 1997Citation ; Lyons-Weiler 1999Citation ), we found that budding yeast CAK1 and fission yeast csk1 have significantly high Ftv scores (25.2 and 32.4, respectively, relative to the alpha = 0.05 significance value of 9.8), suggesting the potential to produce long-branch attractions.

The use of gamma-corrected distances can minimize or negate long-branch attraction (Swofford et al. 1996Citation ). However, the RASA program is currently only capable of analyzing unweighted data sets (Lyons-Weiler 1999Citation ), and therefore we could not determine if our use of gamma-corrected distances sufficiently minimized the potential for long-branch attraction. An alternative method to minimize potential long-branch attraction is to employ the theoretical framework of the S-F method of Brinkmann and Philippe (1999)Citation , in which fast-evolving sites are removed from the data set and phylogenetic relationships are determined based on slow-evolving sites. Fast-evolving sites with multiple substitutions that occurred only within a given taxon are essentially devoid of phylogenetic content and can act to obscure the true phylogeny.

To perform the S-F analysis, we created 17 data sets, S1–S17, reflecting the sequential addition of faster-evolving character groups onto the slowest-evolving (S1) data set. RASA analysis indicated that all of the data sets still had significant Ftv scores for CAK1 and csk1 (data not shown). Both CAK1 and csk1 are notable for having changes in sites that are largely invariant. These changes are concentrated in the S1 and S2 data sets, which contain 16 of 34 positions in which either Cak1 or Csk1 have differences relative to an invariant amino acid in the rest of the CDK family taxa. The effect of these differences from a nearly invariant consensus is to make CAK1 and csk1 more dissimilar relative to other taxa and contribute to the potential problem of long-branch attraction. The S1 and S2 groups were removed from the S3–S17 data sets to produce S3-3–S3-17 data sets. Data sets S3-8–S3-17 still had significant CAK1 and csk1 Ftv scores, while data sets S3-3–S3-7 did not have significant Ftv scores for any taxa (data not shown). Data sets S3-4–S3-7 retained significant association between CAK1 and csk1, with bootstrap support values of 90%–99% (fig. 3 ). Data set S3-3 did not have sufficient phylogenetic information to resolve the clades (data not shown). Therefore, the results of both the gamma-corrected phylogenies and the S-F reduced character phylogenies suggest that the CAK1-csk1 association is not due to the effects of long-branch attraction.



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Fig. 3.—Neighbor-joining (NJ) phylogeny of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans, and human CDKs using the S3-7 data set. The S3-7 data set (containing 95 characters) does not have significant Ftv values, suggesting that there are no taxa manifesting long-branch attraction problems. Bootstrap support values above 50% are given at branch nodes and are derived from maximum-likelihood (ML) and NJ analyses (separated by slash marks). Branch lengths are proportional to the estimated number of amino acid substitutions; the scale bar indicates amino acid substitutions per site. Bootstrap support values above 50% are given at branch nodes and are derived from ML and NJ analyses of the S3-7 data set (separated by slash marks). Species are denoted by cartoon. Ancestral clades are denoted by brackets on the right

 
Are there metazoan orthologs of CAK1 and csk1? There are a number of orphan CDK-family members that do not have apparent yeast orthologs, the KKIALRE clade, CDK4/6 clade, D.m. AC014407, C.e. H01G02.2, and H.s. CCRK. Two orphans, D.m. AC014407 and C.e. H01G02.2, branched without significant bootstrap support at the base of CAK1 and csk1 in the ML and NJ analyses, while H.s. CCRK branched nearby (fig. 2 ). One of the hallmarks of Cak1 and Csk1 is that they are activated without the requirement for phosphorylation at Thr 160 of the T-loop. Both Cak1 and Csk1 lack a phosphorylatable serine (Ser, S) or threonine (Thr, T) residue at this site, and instead contain hydrophobic amino acids (fig. 1 ). In contrast, the majority of CDKs contain either a Ser or a Thr at this site. The exceptions are C. elegans H01G02.2 and every member of the SRB10 clade, which contain an aspartic acid (Asp, D) residue at this site, and human CCRK, which has a deletion encompassing most of the T-loop. It is known that placement of an acidic residue (such as aspartic acid) in the place of a Ser or a Thr can often mimic the effect of Ser or Thr phosphorylation; therefore, the aspartic acid may allow constitutive activation in a manner analogous to phosphorylation of the Thr 160 site (Johnson, Noble, and Owen 1996Citation ). Thus, the primary sequences C.e. H01G02.2 and H.s. CCRK suggest that they will be constitutively active, either due to the presence of an acidic amino acid at position 160 or due to the lack of the inhibitory T-loop. In contrast, the CDK family member that branches nearest to CAK1 and csk1 (although without support), D.m. AC014407, has a Ser residue at position 160, suggesting that it will not be constitutively active.

Experiments in our laboratory have failed to support a role for H01G02.2 as a CAK. After inactivating the H01G02.2 gene in C. elegans using double-stranded RNA-mediated interference (RNAi) (Fire et al. 1998Citation ), we observed variable hyperdermal defects that were not interpretable as cell cycle arrest (data not shown). To test for redundancy of CAK function, we inactivated both H01G02.2 and cdk-7 and found that the embryonic arrest phenotype was the same as that for RNAi of cdk-7 alone. Expression of H01G02.2 in two budding yeast cak1 mutants, cak1-22 (Kaldis, Sutton, and Solomon 1996Citation ) and civ1-4 (Thuret et al. 1996Citation ), failed to rescue the lethal cak1 mutant phenotype (data not shown). In contrast, other researchers have expressed csk1 in the same mutants and found that it complemented cak1 (Hermand et al. 1998Citation ). These results suggest that H01G02.2 is not likely to function as a CAK.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We identified 14 CDK family members in C. elegans, 13 in D. melanogaster, 8 in S. pombe, 7 in S. cerevisiae, and 21 in humans. There are 10 CDK clades, of which 7 contain yeast and metazoan members. Two other clades, CDK4/6 and KKIALRE, contain only metazoan members. The CAK1 clade contains the budding yeast and fission yeast CAKs, CAK1 and csk1, which group together with significant bootstrap scores. Our analysis suggests that association of the divergent CDK family members CAK1 and csk1 is not due to the phenomenon of long-branch attraction.

Four of the CDK clades contain members that have been shown to regulate transcription as CTD kinases of RNA Polymerase II. The KIN28 clade contains budding yeast Kin28 and human CDK7, both of which function as part of the general transcription factor TFIIH as CTD kinases to promote transcription (Orphanides, Lagrange, and Reinberg 1996Citation ). Kin28 is required for the transcription of 87% of yeast genes (Holstege et al. 1998Citation ). The SRB10 clade contains budding yeast Srb10 and human CDK8, both of which function as CTD kinases that negatively regulate transcription by phosphorylating the CTD of RNA Pol II prior to the assembly of the preinitiation complex (Hampsey and Reinberg 1999Citation ). Srb10 negatively regulates 3% of yeast genes (Holstege et al. 1998Citation ). The SGV1 clade contains H.s. CDK9 and D.m. Cdk9, both of which function as CTD kinases in pTEFb (positive transcription elongation factor b) to promote productive RNA Pol II elongation (Zhu et al. 1997Citation ; Reines, Conaway, and Conaway 1999Citation ). An sgv1 mutant has been found to suppress the effects of a deletion of an upstream activating sequence in yeast, suggesting a function in transcriptional regulation, but the exact mechanism of SGV1 action has not been determined (Prelich and Winston 1993Citation ). The CTK1 clade contains budding yeast CTK1, which was isolated as an in vitro CTD kinase (Lee and Greenleaf 1991Citation ). CTK1 was subsequently found to promote RNA Pol II elongation in vitro (Lee and Greenleaf 1997Citation ) and to both activate and repress a subset of yeast genes in vivo (Patturajan et al. 1999Citation ). It is interesting that of the seven ancestral CDK clades with both yeast and metazoan members, only the four CTD kinase-containing clades did not have an expansion in gene number from yeast to metazoa. In each of these clades, there is only a single ortholog in each species, with the exception of two closely related human genes in the CTK1 clade, CHED and KIAA0904.

The number of CDKs that regulate cell cycle progression has increased considerably from yeast to metazoa. In both budding and fission yeast, a single CDK, Cdc28 or Cdc2, respectively, is largely responsible for initiating all cell cycle progressions. In metazoans, H.s. CDK1/C.e. NCC-1/D.m. Cdc2 function in mitotic and meiotic progression; H.s. CDK4/H.s. CDK6/C.e. CDK-4/D.m. Cdk4 regulate G1 phase progression; and H.s. CDK2/D.m. Cdc2c function in S phase progression (King, Jackson, and Kirschner 1994Citation ; Sherr 1994Citation ; Stillman 1996Citation ; Follette and O'Farrell 1997Citation ; Boxem, Srinivasan, and van den Heuvel 1999Citation ; Park and Krause 1999Citation ). In contrast to this conservation of metazoan cell cycle CDKs, there is not a definitive ortholog of the S phase CDK, CDK2, in C. elegans. A potential C. elegans CDK2 counterpart is K03E5.3. While K03E5.3 branched at the base of the CDC28 and PHO85 clades (fig. 2 ), distance matrices show that K03E5.3 is most similar to CDK2 and CDK3 (table 3 ). It will be interesting to determine if K03E5.3 is required for DNA replication in C. elegans.


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Table 3 JTT Distance Matrix for Potential Caenorhabditis elegans G1 and S Phase CDKs

 
The CDK4/CDK6 clade branches from the base of the CDC28 and PHO85 clades (fig. 2 ). Our analysis does not allow us to determine whether the ancestral founder of the CDK4/CDK6 clade was the CDC28 ancestor or the PHO85 ancestor. Members of the CDK4/CDK6 clade are more similar on average to members of the CDC28 clade than to members of the PHO85 clade (table 2 ; data not shown). While Cdc28 is the primary CDK for G1 phase progression in budding yeast, Pho85 has a supporting role facilitating G1 phase progression, and therefore it is possible to envision a conservation of function from either ancestral CDK.

The PHO85 clade has also undergone expansion in metazoans. There are three major metazoan subbranches, the CDK5 branch, the PFTAIRE branch, and the PCTAIRE branch (fig. 2 ). There is a single orthologous gene in the CDK5 branch for all three metazoans. In vertebrates, CDK5 is expressed in postmitotic neuronal cells and is required for central nervous system and brain development (Ohshima et al. 1996Citation ; Chae et al. 1997Citation ). The PFTAIRE branch also has a single orthologous gene in all three metazoans. The function of the H.s. PFTK1 is unknown; its murine ortholog, PFTAIRE-1, is expressed in brain, testes, and embryos (Besset, Rhee, and Wolgemuth 1998Citation ). There are three PCTAIRE subfamily members in humans and one in C. elegans, PCTAIRE1, 2, and 3 and pct-1, respectively. Noticeably absent is a D. melanogaster PCTAIRE family member. The three human PCTAIRE proteins have a high percentage of sequence identity among themselves (82%–85%), suggesting a recent gene duplication. The functions of the human or C. elegans PCTAIRE genes are currently unknown.

The BC18H10.15 clade is unique as an ancestral clade that contains a fission yeast CDK family member, BC18H10.15, but no budding yeast counterpart. This presumably occurred due to the loss of the ancestral gene in budding yeast after the evolutionary divergence of yeast and metazoans. The BC18H10.15 clade contains two subbranches, PISSLRE and PITSLRE. The PISSLRE branch contains H.s. PISSLRE and D.m. Dcdrk. The PITSLRE branch contains two tandemly repeated human PITSLRE genes, two C. elegans genes, B0495.2 and ZC504.3, and D.m. Pitslre. The two expressed human PITSLRE genes, A and B, have only four amino acid differences in the kinase domain and are processed by alternative splicing into multiple isoforms (Lahti, Xiang, and Kidd 1995Citation ). PITSLRE proteins are proteolytically processed during apoptosis to produce active truncated proteins (Lahti, Xiang, and Kidd 1995Citation ). Expression of a truncated PITSLRE, similar to the proteolytically processed form, can induce apoptosis, and cells lacking PITSLRE have been found to be defective for apoptosis, suggesting that PITSLRE is an integral component of the apoptotic pathway (Lahti et al. 1995Citation ; Ariza et al. 1999Citation ). The functions of the ubiquitously expressed, nonproteolytically processed forms of PITSLRE are unknown. Inactivation of PISSLRE in mammalian cells with antisense and dominant-negative constructs produced a G2/M phase cell cycle arrest, suggesting a requirement of PISSLRE in this cell cycle transition (Li et al. 1995Citation ).

The KKIALRE clade is an orphan clade that contains three human genes, KKIALRE, KKIAMRE, and STK9, one C. elegans gene, and one D. melanogaster gene. The cellular functions of none of these genes have been determined.

CAK1/csk1
Experimental data indicate that CAK1 and csk1 have analogous functions (Hermand et al. 1998Citation ; Lee et al. 1999Citation ). Our phylogenetic analysis indicates that they are orthologs. While the two taxa are very divergent, we believe that their branching together is indicative of a real orthologous relationship for three reasons. First, we observed significant CAK1-csk1 association with both the ML and the NJ methods, which are relatively resistant to the effects of long-branch attraction (Felsenstein 1978Citation ; Saitou and Imanishi 1989Citation ). Furthermore, we included a comprehensive data set of all available yeast, C. elegans, D. melanogaster, and human CDKs, as well as a variety of the closest outgroup taxa to minimize potential long-branch attraction effects (Lyons-Weiler and Hoelzer 1997Citation ; Philippe and Laurent 1998Citation ). Second, the CAK1/csk1 association remained after gamma rate correction of the data set to incorporate unequal rates of evolution at amino acid sites. Third, a reduced data set that no longer had a significant RASA Ftv (indicating the absence or diminution of a long-branch attraction problem) still had significant association of CAK1 and csk1.

The finding of orthologous CAK1/csk1 genes in both budding and fission yeast, which are distantly related to each other, suggests that an ortholog existed in the common ancestor of all eukaryotes, although it cannot be excluded that CAK1/csk1 arose in a fungal ancestor. In both NJ and ML analyses, the metazoan CDK family members D.m. AC014407 and C.e. H01G02.2 branched at the base of the CAK1 and csk1 node, while the human gene CCRK branched nearby. These associations were not well supported, with bootstrap values of between 17% and 27%. There is also no significant similarity between the primary sequence of these genes and CAK1 or csk1 (table 2 ; data not shown). Finally, there is no compelling evidence for orthology among the three metazoan genes themselves, which do not associate with high support values (fig. 2 ). Therefore, while it is possible that these genes arose from a CAK1/csk1 ancestor, sequence analysis cannot ascertain orthology. It is interesting that the primary sequence of C.e. H01G02.2 and H.s. CCRK indicates that they may be constitutively active. However, RNAi analysis suggests that C.e. H01G02.2 will not function as a CAK. It will be interesting to learn whether D.m. AC014407 or H.s. CCRK have CAK activity.

The absence of clearly identifiable CAK1/csk1 orthologs in metazoans lends support to a model for CAK function in metazoans originally proposed by Fisher et al. (1995)Citation (discussed below) and suggests a model for the evolution of CAK regulation in which the CAK1/csk1 gene was lost in metazoans. The model envisions an evolutionary progression from an ancestral state that was similar to the current situation in budding yeast, in which CAK1/csk1 provides the only CAK activity for all CDKs (fig. 4 ). In a later ancestor, similar to fission yeast, the transcription CDK, Mcs6/CDK7, acquired the ability to function as a CAK and functions redundantly with Cak1/Csk1 to activate other CDKs. Although redundant for CAK activity, neither Cak1/Csk1 nor Mcs6/CDK7 is in danger of being eliminated, as Cak1/Csk1 is required to activate Mcs6/CDK7 and Mcs6/CDK7 is an essential transcription component. The final evolutionary progression is to a state similar to metazoans in which CAK1/csk1 has been eliminated. To allow this elimination, an alternate CAK for Mcs6/CDK7 is necessary. It has been observed that CDK2-Cyclin A and CDC2-Cyclin B can phosphorylate CDK7 in vitro (Fisher et al. 1995Citation ; Martinez et al. 1997Citation ) and therefore could activate CDK7 in vivo. However, this leaves a theoretical quandary, in that for CDK7 to become active, it must first activate its own CAK. A potential solution, proposed by Fisher et al. (1995)Citation , is that CDK7 can be activated by binding to the assembly factor MAT1 (Fisher et al. 1995Citation ). MAT1 facilitates interaction between CDK7 and Cyclin H, and the trimeric complex is active without Thr170 phosphorylation. In higher eukaryotes, MAT1 is found in complex with CDK7 both within and outside of the TFIIH transcription complex (Fisher et al. 1995Citation ). In contrast, in budding yeast, MAT1 is found solely within the transcription complex (Feaver et al. 1997Citation ), indicating that it would not be available to activate Kin28. The model suggests that the ability of MAT1 to form a complex with Mcs6/CDK7 independently of the TFIIH complex made Cak1/Csk1 activity completely redundant and therefore allowed its loss during evolution. Much of this model remains untested; in particular, it is not known if (1) other CDKs function as CAKs for CDK7 in vivo, (2) the MAT1/CDK7/Cyclin H complex functions as a CAK in vivo, or (3) CDK7 is the CAK for all CDKs in metazoans.



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Fig. 4.—Model of CAK evolution in yeast and metazoans. Evolutionary progression is shown, starting with the CAK regulation now found in budding yeast (left), in which Cak1 phosphorylates and activates the cell cycle CDK (Cdc28) and the transcription CDK (Kin28). The next progression is to the CAK regulation now found in fission yeast (center), in which the Cak1 ortholog, Csk1, activates the cell cycle CDK (Cdc2) and the transcription CDK (Mcs6). Mcs6 also has CAK activity and redundantly activates Cdc2. The final progression is to the CAK regulation now found in metazoa (right), in which there is no Cak1/Csk1 ortholog. The transcription CDK, CDK7, is activated by phosphorylation by cell cycle CDKs. CDK7 in combination with MAT1 and Cyclin H is capable of being active without Thr 160 phosphorylation and this combination is capable of acting as a CAK to phosphorylate other CDKs

 



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Fig. 1 (Continued)

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank Christian Zmasek, Ann E. Sluder, and Russell L. Malmberg for helpful discussions and critical reading of the manuscript; Carl Mann, Philipp Kaldis, and Mark J. Solomon for yeast strains; and Claiborne V. Glover and Sricharan Bandhakavi for yeast and Drosophila reagents. This work was supported by NIH grant GM55297 and HFSPO grant RG-229/98 to E.T.K.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Keywords: cyclin dependent kinase evolution CDK activating kinase long-branch attraction CDK CAK Back

2 Address for correspondence and reprints: Edward T. Kipreos, Department of Cellular Biology, University of Georgia, Athens, Georgia 30602. E-mail: ekipreos{at}cb.uga.edu Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

    Adachi, J., and M. Hasegawa. 1996. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28:1–150.

    Adams, M. D., S. E. Celniker, R. A. Holt et al. (195 co-authors). 2000. The genome sequence of Drosophila melanogaster. Science 287:2185–2195.

    Altschul, S. F., T. L. Madden, A. A. Schaffer, J. H. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402.[Abstract/Free Full Text]

    Ariza, M. E., M. Broome-Powell, J. M. Lahti, V. J. Kidd, and M. A. Nelson. 1999. Fas-induced apoptosis in human malignant melanoma cell lines is associated with the activation of the p34(cdc2)-related PITSLRE protein kinases. J. Biol. Chem. 274:28505–28513.[Abstract/Free Full Text]

    Beach, D., B. Durkacz, and P. Nurse. 1982. Functionally homologous cell cycle control genes in budding and fission yeast. Nature 300:706–709.

    Besset, V., K. Rhee, and D. J. Wolgemuth. 1998. The identification and characterization of expression of Pftaire-1, a novel Cdk family member, suggest its function in the mouse testis and nervous system. Mol. Reprod. Dev. 50:18–29.[ISI][Medline]

    Boxem, M., D. G. Srinivasan, and S. van den Heuvel. 1999. The Caenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase required for M phase in meiotic and mitotic cell divisions, but not for S phase. Development 126:2227–2239.

    Brinkmann, H., and H. Philippe. 1999. Archaea sister group of Bacteria? Indications from tree reconstruction artifacts in ancient phylogenies. Mol. Biol. Evol. 16:817–825.[Abstract]

    Buck, V., P. Russell, and J. B. Millar. 1995. Identification of a cdk-activating kinase in fission yeast. EMBO J. 14:6173–6183.[Abstract]

    The C. ELEGANS Sequencing Consortium. (517 co-authors). 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018.

    Chae, T., Y. T. Kwon, R. Bronson, P. Dikkes, E. Li, and L. H. Tsai. 1997. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18:29–42.

    Cismowski, M. J., G. M. Laff, M. J. Solomon, and S. I. Reed. 1995. 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. 15:2983–2992.[Abstract]

    Damagnez, V., T. P. Makela, and G. Cottarel. 1995. Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. EMBO J. 14:6164–6172.[Abstract]

    Darbon, J. M., A. Devault, S. Taviaux, D. Fesquet, A. M. Martinez, S. Galas, J. C. Cavadore, M. Doree, and J. M. Blanchard. 1994. Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of cdk-activating kinase. Oncogene 9:3127–3138.

    De Bondt, H. L., J. Rosenblatt, J. Jancarik, H. D. Jones, D. O. Morgan, and S. H. Kim. 1993. Crystal structure of cyclin-dependent kinase 2. Nature 363:595–602.

    Espinoza, F. H., A. Farrell, H. Erdjument-Bromage, P. Tempst, and D. O. Morgan. 1996. A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK. Science 273:1714–1717.

    Espinoza, F. H., A. Farrell, J. L. Nourse, H. M. Chamberlin, O. Gileadi, and D. O. Morgan. 1998. Cak1 is required for Kin28 phosphorylation and activation in vivo. Mol. Cell. Biol. 18:6365–6373.[Abstract/Free Full Text]

    Espinoza, F. H., J. Ogas, I. Hershowitz, and D. O. Morgan. 1994. Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85. Science 266:1388–1391.

    Feaver, W. J., N. L. Henry, Z. Wang, X. Wu, J. Q. Svejstrup, D. A. Bushnell, E. C. Friedberg, and R. D. Kornberg. 1997. Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits. J. Biol. Chem. 272:19319–19327.[Abstract/Free Full Text]

    Feaver, W. J., J. Q. Svejstrup, N. L. Henry, and R. D. Kornberg. 1994. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell 79:1103–1109.

    Felsenstein, J. 1978. Cases in which parsimony and compatibility methods will be positively misleading. Syst. Zool. 24:401–410.

    ———. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:368–376.[ISI][Medline]

    ———. 1993. PHYLIP (phylogeny inference package). Version 3.5c. Distributed by the author, Department of Genetics, University of Washington, Seattle.

    Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811.

    Fisher, R. P., P. Jin, H. M. Chamberlin, and D. O. Morgan. 1995. Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. Cell 83:47–57.

    Follette, P. J., and P. H. O'Farrell. 1997. Cdks and the Drosophila cell cycle. Curr. Opin. Genet. Dev. 7:17–22.[ISI][Medline]

    Gribskov, M., R. Luthy, and D. Eisenberg. 1990. Profile analysis. Methods Enzymol. 183:146–159.[ISI][Medline]

    Hampsey, M., and D. Reinberg. 1999. RNA polymerase II as a control panel for multiple coactivator complexes. Curr. Opin. Genet. Dev. 9:132–139.[ISI][Medline]

    Hanks, S. K., and T. Hunter. 1995. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9:576–596.[Abstract/Free Full Text]

    Hermand, D., A. Pihlak, T. Westerling, V. Damagnez, J. Vandenhaute, G. Cottarel, and T. P. Makela. 1998. Fission yeast Csk1 is a CAK-activating kinase (CAKAK). EMBO J. 17:7230–7238.[Abstract/Free Full Text]

    Hindley, J., and G. A. Phear. 1984. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene 31:129–134.

    Holstege, F. C., E. G. Jennings, J. J. Wyrick, T. I. Lee, C. J. Hengartner, M. R. Green, T. R. Golub, E. S. Lander, and R. A. Young. 1998. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95:717–728.

    Johnson, L. N., M. E. Noble, and D. J. Owen. 1996. Active and inactive protein kinases: structural basis for regulation. Cell 85:149–158.

    Kaldis, P. 1999. The cdk-activating kinase (CAK): from yeast to mammals. Cell. Mol. Life Sci. 55:284–296.[ISI][Medline]

    Kaldis, P., A. Sutton, and M. J. Solomon. 1996. The Cdk-activating kinase (CAK) from budding yeast. Cell 86:553–564.

    King, R. W., P. K. Jackson, and M. W. Kirschner. 1994. Mitosis in transition. Cell 79:563–571.

    Lahti, J. M., J. Xiang, L. S. Heath, D. Campana, and V. J. Kidd. 1995. PITSLRE protein kinase activity is associated with apoptosis. Mol. Cell. Biol. 15:1–11.[Abstract]

    Lahti, J. M., J. Xiang, and V. J. Kidd. 1995. The PITSLRE protein kinase family. Prog. Cell Cycle Res. 1:329–338.[Medline]

    Larochelle, S., J. Pandur, R. P. Fisher, H. K. Salz, and B. Suter. 1998. Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes Dev. 12:370–381.[Abstract/Free Full Text]

    Lee, J., and A. L. Greenleaf. 1991. CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. Gene Expr. 1:149–167.[ISI][Medline]

    ———. 1997. Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I. J. Biol. Chem. 272:10990–10993.[Abstract/Free Full Text]

    Lee, K. M., J. E. Saiz, W. A. Barton, and R. P. Fisher. 1999. Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs). Curr. Biol. 9:441–444.[ISI][Medline]

    Lenburg, M. E., and E. K. O'Shea. 1996. Signaling phosphate starvation. Trends Biochem. Sci. 21:383–387.[ISI][Medline]

    Li, S., T. K. Maclachlan, A. De Luca, P. P. Claudio, G. Condorelli, and A. Giordano. 1995. The cdc-2-related kinase, PISSLRE, is essential for cell growth and acts in G2 phase of the cell cycle. Cancer Res. 55:3992–3995.[Abstract]

    Liao, S. M., J. Zhang, D. A. Jeffery, A. J. Koleske, C. M. Thompson, D. M. Chao, M. Viljoen, H. J. Van Vuuren, and R. A. Young. 1995. A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374:193–196.

    Lorincz, A. T., and S. I. Reed. 1984. Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature 307:183–185.

    Lyons-Weiler, J. F. 1999. RASA 2.3 software and documentation for the Mac. http://test1.bio.psu.edu/LW/rasatext.html.

    Lyons-Weiler, J., and G. A. Hoelzer. 1997. Escaping from the Felsenstein zone by detecting long branches in phylogenetic data. Mol. Phylogenet. Evol. 8:375–384.[ISI][Medline]

    Lyons-Weiler, J., G. A. Hoelzer, and R. J. Tausch. 1996. Relative apparent synapomorphy analysis (RASA). I: the statistical measurement of phylogenetic signal. Mol. Biol. Evol. 13:749–757.[Abstract]

    Martinez, A. M., M. Afshar, F. Martin, J. C. Cavadore, J. C. Labbe, and M. Doree. 1997. Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity. EMBO J. 16:343–354.[Abstract/Free Full Text]

    Measday, V., L. Moore, J. Ogdas, M. Tyers, and B. Andrews. 1994. The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex: a cell cycle regulator in yeast. Science 266:1391–1395.

    Molz, L., and D. Beach. 1993. Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity. EMBO J. 12:1723–1732.[Abstract]

    Morgan, D. O. 1997. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol. 13:261.

    Myerson, M., G. H. Enders, C.-L. Wu, L.-K. Su, C. Gorka, C. Nelson, E. Harlow, and L.-H. Tsai. 1992. A family of human cdc2-related protein kinases. EMBO J. 11:2909–2917.[Abstract]

    Nasmyth, K. A., and S. I. Reed. 1980. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA 77:2119–2123.

    Ohshima, T., J. M. Ward, C. G. Huh, G. Longenecker, Veeranna, H. C. Pant, R. O. Brady, L. J. Martin, and A. B. Kulkarni. 1996. Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc. Natl. Acad. Sci. USA 93:11173–11178.

    Orphanides, G., T. Lagrange, and D. Reinberg. 1996. The general transcription factors of RNA polymerase II. Genes Dev. 10:2657–2683.[ISI][Medline]

    Park, M., and M. W. Krause. 1999. Regulation of postembryonic G(1) cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex. Development 126:4849–4860.

    Patturajan, M., N. K. Conrad, D. B. Bregman, and J. L. Corden. 1999. Yeast carboxyl-terminal domain kinase I positively and negatively regulates RNA polymerase II carboxyl-terminal domain phosphorylation. J. Biol. Chem. 274:27823–27828.[Abstract/Free Full Text]

    Philippe, H., and J. Laurent. 1998. How good are deep phylogenetic trees? Curr. Opin. Genet. Dev. 8:616–623.[ISI][Medline]

    Pines, J. 1995. Cyclins and cyclin-dependent kinases: a biochemical view. Biochem. J. 308:697–711.[ISI][Medline]

    Poon, R. Y. C., K. Yamashita, J. P. Adamczewski, T. Hunt, and J. Shuttleworth. 1993. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO J. 12:3123–3132.[Abstract]

    Prelich, G., and F. Winston. 1993. Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo. Genetics 135:665–676.

    Reines, D., R. C. Conaway, and J. W. Conaway. 1999. Mechanism and regulation of transcriptional elongation by RNA polymerase II. Curr. Opin. Cell Biol. 11:342–346.[ISI][Medline]

    Russo, A. A., P. D. Jeffrey, and N. P. Pavletich. 1996. Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat. Struct. Biol. 3:696–700.[ISI][Medline]

    Saitou, N., and T. Imanishi. 1989. Relative efficiencies of the Fitch-Margoliash, maximum parsimony, maximum-likelihood, minimum-evolution, and neighbor-joining methods of phylogenetic tree construction in obtaining the correct tree. Mol. Biol. Evol. 6:514–525.[Free Full Text]

    Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.[Abstract]

    Sherr, C. J. 1994. G1 phase progression: cycling on cue. Cell 79:551–555.

    Solomon, M. J., J. W. Harper, and J. Shuttleworth. 1993. CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40mo15. EMBO J. 12:3133–3142.[Abstract]

    Sterner, D. E., J. M. Lee, S. E. Hardin, and A. L. Greenleaf. 1995. The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex. Mol. Cell. Biol. 15:5716–5724.[Abstract]

    Stillman, B. 1996. Cell cycle control of DNA replication. Science 274:1659–1664.

    Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964–969.[Free Full Text]

    Swofford, D. L. 1993. PAUP: phylogenetic analysis using parsimony. Version 3.1. Illinois Natural History Survey, Champaign.

    Swofford, D. L., G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. Pp. 407–514 in D. M. Hillis, C. Moritz, and B. K. Mable, eds. Molecular systematics, Sinauer, Sunderland, Mass.

    Tassan, J. P., S. J. Schultz, J. Bartek, and E. A. Nigg. 1994. Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). J. Cell Biol. 127:467–478.[Abstract]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.[Abstract]

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

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

    van den Heuvel, S., and E. Harlow. 1993. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262:2050–2054.

    Yang, Z. 1996. Among site rate variation and its impact on phylogenetic analyses. Trends Ecol. Evol. 11:367–372.[ISI]

    Zhu, Y., T. Pe'ery, J. Peng, Y. Ramanathan, N. Marshall, T. Marshall, B. Amendt, M. B. Mathews, and D. H. Price. 1997. Transcription elongation factor P-TEFb is required for HIV-1 Tat transactivation in vitro. Genes Dev. 11:2622–2632.[Abstract/Free Full Text]

Accepted for publication April 5, 2000.