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| Milestone 19
Cyclin' into S phase
Progression through the cell cycle is highly regulated. In all eukaryotic cells, entry into M phase is regulated by a complex of the cyclin-dependent kinase (CDK) cdc2 and cyclin B (also known as MPF; see milestone 7). Entry into S phase is regulated in a similar way in both fission and budding yeast. But in 1991, a different mechanism was discovered in vertebrates, leading to the identification of a new class of proteins.
In the fission yeast Schizosaccharomyces pombe, cdc2 is not only involved in the progression into M phase, but also in progression into S phase. The budding yeast orthologue, CDC28, is likewise required for entry into both S phase and M phase. The activity of this CDK is regulated by cyclins at both of these checkpoints in budding and fission yeast. In 1991, five groups independently reported the identification of a new class of cyclins, and two other groups identified a cdc2-related kinase, CDK2. Together, CDK2 and these new cyclins regulate the G1/S checkpoint in vertebrates. The laboratories of Chuck Sherr, David Beach, Steve Reed, Jim Roberts and Andrew Arnold simultaneously identified a new class of cyclins — which consists of cyclins C, D and E — in mammals, using a variety of techniques. Sherr and colleagues used a subtractive hybridization approach to screen for genes that are induced by colony-stimulating factor ( CSF-1), a growth factor that is required for macrophage progression through the G1/S boundary. The groups of Beach, Reed and Roberts complemented yeast strains that do not express CLN1 and CLN2, and are conditionally defective in CLN3 expression, by transfection of cDNAs from a human library. Arnold and colleagues used a probe from the locus of a candidate oncogene to hybridize a human placental cDNA library at low stringency to identify the gene. The genes that encode cyclins C, D and E show clear homology to yeast cyclins and to the genes that encode vertebrate cyclin A and B. However, cyclin D and cyclin E are clearly part of a separate family, as they are more similar to one another than to the genes that encode cyclin A or B. They also share the same exon-intron boundaries. In addition, cyclins A and B bear a so-called 'destruction box', which is involved in regulating their degradation after M-phase entry. Cyclins C, D and E do not have that sequence; instead, their sequence shows a PEST sequence that is also involved in regulating protein stability. The relevant catalytic subunit of this 'S-phase factor' was cloned independently in parallel studies from Steve Elledge's and Ed Harlow's groups. The first group complemented a cdc28-deficient budding yeast strain with the human cDNA that encodes CDK2. The second group cloned the cdc2-related gene by degenerate PCR amplification and hybridization under conditions of low stringency. Like its cdc2 homologue, CDK2 binds cyclins, which regulate its activity; it is also regulated by phosphorylation. It was later established that cyclin E is — together with cyclin A - the regulatory subunit for the CDK2 kinase in S phase. It was also determined that cyclin D functions as a regulator of CDK4, and CDK6 in G1, and in the regulation of the expression of cyclin E. However, ten years after the publication of this work, the exact function of cyclin C has yet to be established.
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