CDK inhibitory proteins
Association with small inhibitory proteins is a universal mechanism of CDK regulation (Sherr and Roberts, 1999), though the CKIs (Cyclin-dependent Kinase Inhibitors) involved are highly divergent between yeasts and metazoans. Three different proteins, p21Cip1, p27Kip1 and p57Kip2, form the "CDK inhibitory Protein/Kinase Inhibitor protein" (Cip/Kip) family in mammals. The C. elegans genome encodes two members of this family: CKI-1 and CKI-2 (Feng et al., 1999; Hong et al., 1998). Although both predicted proteins are similarly close in amino-acid sequence to p21Cip1 and p27Kip1, only CKI-1 appears to act generally in cell-cycle control (Boxem and van den Heuvel, 2001; Feng et al., 1999; Fukuyama et al., 2003; Hong et al., 1998).
Several results have shown that CKI-1 acts to promote cell-cycle arrest throughout development, analogous to p27Kip1 in mammals and Dacapo in flies. Absence of cki-1 as a result of the mnDf100 deletion results in embryonic arrest with hyperplasia in multiple lineages, including the intestinal and hypodermal lineages, as well as increased apoptosis and defects in morphogenesis (Fukuyama et al., 2003). mnDf100 also eliminates cki-2 and other genes, but cki-1 genomic sequences partly suppress the phenotype. Moreover, similar defects were observed in a fraction of cki-1(RNAi) embryos. However, RNAi usually causes incomplete inactivation of cki-1 and predominantly gives rise to sterile adults with extra divisions in the postembryonic lineages and extra gonad arms (Shiva phenotype; Hong et al., 1998). Postembryonic precursor cells in such cki-1(RNAi) animals fail to arrest in G1 and ectopically express the S phase marker rnr::gfp. Thus, cki-1 Kip1 function is rate limiting for S phase entry, particularly in cells that enter a prolonged quiescent state before re-entering the cell cycle.
Interestingly, loss of cki-1 Kip1 also affects aspects of cell-fate determination, as the extra distal tip cells (DTCs) in cki-1(RNAi) animals are derived from a different cell type and not from DTC duplication (Kostic et al., 2003).
Several results have shown that CKI-1 acts to promote cell-cycle arrest throughout development, analogous to p27Kip1 in mammals and Dacapo in flies. Absence of cki-1 as a result of the mnDf100 deletion results in embryonic arrest with hyperplasia in multiple lineages, including the intestinal and hypodermal lineages, as well as increased apoptosis and defects in morphogenesis (Fukuyama et al., 2003). mnDf100 also eliminates cki-2 and other genes, but cki-1 genomic sequences partly suppress the phenotype. Moreover, similar defects were observed in a fraction of cki-1(RNAi) embryos. However, RNAi usually causes incomplete inactivation of cki-1 and predominantly gives rise to sterile adults with extra divisions in the postembryonic lineages and extra gonad arms (Shiva phenotype; Hong et al., 1998). Postembryonic precursor cells in such cki-1(RNAi) animals fail to arrest in G1 and ectopically express the S phase marker rnr::gfp. Thus, cki-1 Kip1 function is rate limiting for S phase entry, particularly in cells that enter a prolonged quiescent state before re-entering the cell cycle.
Interestingly, loss of cki-1 Kip1 also affects aspects of cell-fate determination, as the extra distal tip cells (DTCs) in cki-1(RNAi) animals are derived from a different cell type and not from DTC duplication (Kostic et al., 2003).
The tumor suppressor pRb is a well-known transcriptional repressor of, among others, genes involved in S phase progression such as cyclin E. Proteins of the pRb family exert this role through association with transcription factors, primarily with E2F/DP heterodimers (together referred to as "E2F"; Stevaux and Dyson, 2002). This association prevents "activating E2Fs" from promoting transcription. In addition, to inhibit transcription, "repressive E2Fs" recruit pRb family members and associated chromatin remodeling complexes such as the Nucleosome Remodeling and Deacetylase (NuRD) complex.
Several members of the Rb/E2F pathway in C. elegans have been identified as class B synthetic Multivulva (synMuv) genes (see Vulval development). As such, lin-35 Rb, efl-1E2F4/5, dpl-1 DP and several putative NuRD-complex components all act in a redundant pathway to antagonize Ras-mediated induction of the vulval cell fate. The fact that these genes have similar, rather than opposite, loss-of-function phenotypes provided strong in vivo evidence that E2F/DP can act as a transcriptional repressor, in concert with pRb and NuRD. The contribution of lin-35 Rb in vulval precursor cell (VPC) determination is non cell-autonomous (Myers and Greenwald, 2005), and cannot be explained by lack of cell-cycle control in these cells.
Homozygous lin-35 Rb mutants do not display a prominent increase in cell division, although a fraction of such animals form extra intestinal nuclei (Saito et al., 2004). However, the contribution of lin-35 Rb to negative regulation of G1 progression became apparent in double mutant combinations. Inactivation of lin-35 substantially rescues the larval arrest of cell division in mutants that lack the positive G1 regulators cyd-1 and cdk-4 (Boxem and van den Heuvel, 2001). In addition, lin-35 inactivation synergistically increases the number of extra cell divisions when combined with negative G1 regulators, such as cki-1 Cip/Kip, cdc-14 Cdc14 and fzr-1 Cdh1/FZR (Boxem and van den Heuvel, 2001; Fay et al., 2002; Saito et al., 2004). Together, such results indicate that lin-35 acts redundantly to inhibit G1 progression, likely downstream of cyd-1 and cdk-1 and parallel of cki-1, fzr-1 and cdc-14.
Examination of additional double mutant combinations revealed that some of the other synMuv class B genes also contribute to G1 control (Boxem and van den Heuvel, 2002; Fay et al., 2002). Specifically, efl-1 E2F negatively regulates cell-cycle entry, while dpl-1 DP appears to act both as a positive and negative regulator. In addition, a negative G1regulatory function was identified for lin-9 Mip130/TWIT, as well as lin-15B and lin-36, which encode novel proteins. Class A synMuv genes and class B genes that encode NURD components have not been observed to affect the cell cycle.
Candidate targets of Rb/E2F regulation in C. elegans include cye-1 Cyclin E and rnr-1, which encodes the ribonucleotide reductase large subunit. These genes have multiple E2F-binding sites within their promoter regions (Brodigan et al., 2003; Hong et al., 1998) and are regulated by E2F in other species. Several genetic observations are also consistent with lin-35 acting upstream of cye-1 to repress its transcription (Boxem and van den Heuvel, 2001; our unpublished observations).
Several members of the Rb/E2F pathway in C. elegans have been identified as class B synthetic Multivulva (synMuv) genes (see Vulval development). As such, lin-35 Rb, efl-1E2F4/5, dpl-1 DP and several putative NuRD-complex components all act in a redundant pathway to antagonize Ras-mediated induction of the vulval cell fate. The fact that these genes have similar, rather than opposite, loss-of-function phenotypes provided strong in vivo evidence that E2F/DP can act as a transcriptional repressor, in concert with pRb and NuRD. The contribution of lin-35 Rb in vulval precursor cell (VPC) determination is non cell-autonomous (Myers and Greenwald, 2005), and cannot be explained by lack of cell-cycle control in these cells.
Homozygous lin-35 Rb mutants do not display a prominent increase in cell division, although a fraction of such animals form extra intestinal nuclei (Saito et al., 2004). However, the contribution of lin-35 Rb to negative regulation of G1 progression became apparent in double mutant combinations. Inactivation of lin-35 substantially rescues the larval arrest of cell division in mutants that lack the positive G1 regulators cyd-1 and cdk-4 (Boxem and van den Heuvel, 2001). In addition, lin-35 inactivation synergistically increases the number of extra cell divisions when combined with negative G1 regulators, such as cki-1 Cip/Kip, cdc-14 Cdc14 and fzr-1 Cdh1/FZR (Boxem and van den Heuvel, 2001; Fay et al., 2002; Saito et al., 2004). Together, such results indicate that lin-35 acts redundantly to inhibit G1 progression, likely downstream of cyd-1 and cdk-1 and parallel of cki-1, fzr-1 and cdc-14.
Examination of additional double mutant combinations revealed that some of the other synMuv class B genes also contribute to G1 control (Boxem and van den Heuvel, 2002; Fay et al., 2002). Specifically, efl-1 E2F negatively regulates cell-cycle entry, while dpl-1 DP appears to act both as a positive and negative regulator. In addition, a negative G1regulatory function was identified for lin-9 Mip130/TWIT, as well as lin-15B and lin-36, which encode novel proteins. Class A synMuv genes and class B genes that encode NURD components have not been observed to affect the cell cycle.
Candidate targets of Rb/E2F regulation in C. elegans include cye-1 Cyclin E and rnr-1, which encodes the ribonucleotide reductase large subunit. These genes have multiple E2F-binding sites within their promoter regions (Brodigan et al., 2003; Hong et al., 1998) and are regulated by E2F in other species. Several genetic observations are also consistent with lin-35 acting upstream of cye-1 to repress its transcription (Boxem and van den Heuvel, 2001; our unpublished observations).
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