Regulators of the cell cycle

The C. elegans genome encodes multiple members of the cyclin-dependent kinase (CDK) family. At least two CDKs, CDK-1 and CDK-4, are essential for cell-cycle progression (Boxem et al., 1999; Boxem and van den Heuvel, 2001; Park and Krause, 1999). These CDKs act at distinct times in the cell cycle and use specific cyclin partners, similar to their mammalian orthologs (Table 1).

CDK-1, previously known as NCC-1 for nematode cell cycle, was identified based on its close similarity to the prototypical yeast CDK (Mori et al., 1994). In contrast to yeast, but similar to mammalian Cdk1, cdk-1/(ncc-1) is specifically required for G₂/M progression and not for G₁ or S phase (Boxem et al., 1999). Maternal cdk-1 product suffices for embryogenesis, and candidate null mutant animals arrest cell division during L1 development. Several observations indicate that the post-embryonic precursor cells in these mutants arrest in G₂ phase: such cells show normal expression of the rnr::GFP reporter and BrdU incorporation during S phase, but fail to proceed into mitosis (as indicated by absence of chromosome condensation and nuclear envelope breakdown). Moreover, endoreduplication cycles, which skip M phase, continue in cdk-1 mutants and intestinal nuclei accumulate the normal 32n DNA content. Following RNAi of cdk-1 in adult hermaphrodites, oocytes show delayed meiotic maturation, form an eggshell upon fertilization, but neither align nor segregate homologous chromosomes. Thus, cdk-1 is required for meiotic as well as mitotic M phase.

CDK-1 likely acts with mitotic cyclins of the A and B subfamilies (Kreutzer et al., 1995). A single full-length cyclin A gene (cya-1) is expressed in C. elegans, as well as three typical B-type cyclins (cyb-1, cyb-2.1 and cyb-2.2), and a distinct member of the cyclin B3 subfamily (cyb-3). While cyb-1 and cyb-3 each are individually required during embryonic development, simultaneous inactivation of these mitotic cyclins causes a more severe phenotype: cyb-1;cyb-3(RNAi) embryos arrest at the one-cell stage and resemble cdk-1(RNAi) embryos (our unpublished results). These data support the notion that different mitotic cyclins have functions that are partly distinct and partly overlapping.

The cdk-4 Cdk4/6 kinase and cyd-1 D-type cyclin genes are required for progression through G₁ phase during larval development (Boxem and van den Heuvel, 2001; Park and Krause, 1999). CYD-1 and CDK-4 likely act in complex, as indicated by their direct interaction in vitro and close similarity in null phenotypes (Park and Krause, 1999). In contrast to larval divisions, only a few very late embryonic divisions depend on cyd-1/cdk-4 activity (Boxem and van den Heuvel, 2001; Yanowitz and Fire, 2005). Possible explanations for this difference include that the early embryonic divisions lack a G₁ phase and therefore will not need a G₁ cyclin or CDK. Also, as one of its most important functions, cyd-1 and cdk-4 act to antagonize the transcriptional repressor lin-35 Rb (see below; (Boxem and van den Heuvel, 2001). In the absence of cyd-1/cdk-4 function, lin-35 Rb may inappropriately repress cell-cycle genes, but this cannot prevent divisions that are driven by maternal products. Also, cyd-1 and cdk-4 could primarily promote growth, as in Drosophila (Datar et al., 2000; Meyer et al., 2000), which is not incorporated in the embryonic divisions. However, larval divisions arrest in G₁ while cells continue to grow in the mutants, and growth retardation occurs later (Boxem and van den Heuvel, 2001). Therefore, absence of G₁ phases and maternal contribution of DNA replication components likely explain the limited requirement for cyd-1/cdk-4 during embryogenesis

Taken together, CDK-1 and CDK-4 act in G₂/M and G₁, respectively, like their mammalian orthologs Cdk-1 and Cdk4/6. It is currently not clear whether C. elegans also uses a Cdk2 ortholog, which acts subsequent to Cdk4/6 in mammals to promote G₁/S and S phase progression. The best candidate is K03E5.3, which shares 43% amino-acid identity with human Cdk2 (Boxem et al., 1999). Inhibition of this gene by RNAi resulted in a variable phenotype, with animals arresting during embryogenesis, during early or late larval development, and as sterile adults.

In other metazoans, Cdk2 acts with Cyclin E to promote S phase entry. C. elegans cye-1 Cyclin E deletion animals show surprisingly normal development until the L3 stage, at which time the VPC divisions proceed slowly and incompletely (Fay and Han, 2000). This modest phenotype apparently depends on long lasting maternal function, as RNAi results in embryonic lethality at approximately the hundred-cell stage (Brodigan et al., 2003; Fay and Han, 2000).

Several other members of the Cdk superfamily are present in C. elegans, including a Cdk7/Mo15 ortholog (Boxem et al., 1999; Liu and Kipreos, 2000). Cdk7 was identified as CDK-activating kinase (CAK) in mammalian cells, but also as part of the TFIIH transcription factor, responsible for phosphorylating the C-terminal domain (CTD) of RNA polymerase II (Fisher and Morgan, 1994; Shiekhattar et al., 1995). The combination of a temperature-sensitive mutation and RNAi of cdk-7 resulted in one-cell arrest similar to cdk-1(RNAi) embryos (Wallenfang and Seydoux, 2002). In addition, partial inactivation of cdk-7 interfered with transcription and phosphorylation of the RNA polymerase CTD. These data support dual activities of CDK-7 as both CDK-activating kinase (CAK) and CTD kinase in vivo.

Several other CDKs are likely to act independent of the cell cycle. CDK-5 is remarkably close to human Cdk5, sharing 74% identity at the amino-acid level, which has neuronal functions (Cruz and Tsai, 2004). Two other CDKs, CDK-8 and CDK-9, likely are involved specifically in regulating transcription (Liu and Kipreos, 2000; Shim et al., 2002).