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

Evolution of Cyclin-Dependent Kinases (CDKs) and CDK-Activating Kinases (CAKs): Differential Conservation of CAKs in Yeast and Metazoa Ji Liu and Edward T. Kipreos Department of Cellular Biology, University of Georgia Introduction Cyclin-dependent kinases (CDKs) are serine/threonine kinases that must bind to cyclin proteins to become active (Pines 1995). 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 1994; Sherr 1994; Stillman 1996). 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 1980; Beach, Durkacz, and Nurse 1982; Hindley and Phear 1984; Lorincz and Reed 1984). 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 1997). The functions of many CDKs are still unknown (Morgan 1997). 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 Key words: cyclin dependent kinase, evolution, CDK activating kinase, long-branch attraction, CDK, CAK. Address for correspondence and reprints: Edward T. Kipreos, Department of Cellular Biology, University of Georgia, Athens, Georgia 30602. E-mail: . Mol. Biol. Evol. 17(7):1061–1074. 2000 q 2000 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 (Morgan 1997). 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 1993; King, Jackson, and Kirschner 1994; Sherr 1994; Stillman 1996; Morgan 1997). 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. 1995; Liao et al. 1995; Lee and Greenleaf 1991; Sterner et al. 1995). Sgv1 regulates transcription, potentially also as a CTD kinase, as its ortholog CDK9 functions as a CTD kinase (Prelich and Winston 1993; Reines, Conaway, and Conaway 1999). Finally, Pho85 functions to inhibit gene transcription in response to phosphate (Lenburg and O’Shea 1996). 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. 1994; Measday et al. 1994). 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-activat1061 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. 1062 Liu and Kipreos reported. Since the metazoan CAK CDK7 does not appear to be sufficient for the activation of all CDKs (Larochelle et al. 1998), 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 1998), 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 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 ( (...truncated)