By Levi Clancy for Student Reader on
Cyclin-dependent kinases regulate two important transitions in the eukaryotic cell cycle: entry into S-phase and entry into mitosis. CDKs are controlled at every level: presence (controlled degradation); activity (inhibitors); and even specificity (cyclins). Examples are shown below:
|Cyclins||The specificity of cyclin-dependent kinases is regulated by their cyclins. For example, S. cerevisiae uses the same CDK throughout the entire cell cycle. However, different cyclins bind to the CDK and direct it to different substrates for phosphorylation.|
|Inhibitors||S-cyclin+CDK expression is activated by G1-cyclin+CDK, but S-cyclin+CDK is immediately bound by the inhibitor Sic1. In late G1, G1-cyclin+CDK reaches a high enough concentration to marks Sic1 for degradation. In this manner, S-cyclin+CDK is inactive until the cell is ready for S-phase.|
|Degradation||Mitotic-cylin+CDK (MPF) concentrations peak in metaphase, and its elimination must be very carefully regulated. Mitotic cyclins carry a conserved destruction box, which is bound by APC/C-Cdh1 as soon as all sister chromatids have separated.|
Human CDKs and corresponding Cyclins
|CDK1||cyclin A, cyclin B||CDK2||cyclin A, cyclin E||CDK3||CDK4||cyclin D1, D2, D3|
|CDK5||p35 (a regulator dissimilar to cyclins)||CDK6||cyclin D1, D2, D3||CDK7||cyclin H||CDK8||cyclin C|
|CDK9||cyclin T1, T2a, T2b, cyclin K||CDK10||CDK11||cyclin L|
A cyclin-CDK consists of a regulatory subunit (the cyclin) and a kinase subunit (the cyclin-dependent kinase, aka CDK). CDKs trigger cell cycle events and regulate transcription and mRNA processing (except CDK9, which has a totally unrelated function). CDKs phosphorylate Serine and Threonine residues, but have little or no activity unless bound by a cyclin (hence their name). Cdc28p (the predominant yeast cyclin-dependent kinase involved in cell cycle control) is highly homologous to Cdk2.
|Late G1||Cln1 & Cln2||Cdc28|
|Early S-Phase||Clb5 & Clb6||Cdc28|
|Late S-Phase + Early mitosis||Clb3 & Clb4||Cdc28|
|Late Mitosis||Clb1 & Clb2||Cdc28|
|Mid-G1||Cyclin D's||CDK4 & CDK6|
|Mitotis||Cyclin A &|
|CDK1 & CDK2|
High Copy Suppressor Experiment
Cdc28 (found in budding yeast) is highly homologous to Cdc2, a CDK found in S. pombe (fission yeast). Thus, it was theorized that Cdc28 is a CDK itself; thus, it must have a corresponding cyclin. If Cdc28 is a CDK, then in wild-type cells there must be a G1 cyclin that bound Cdc28 to form an S-phase promoting factor (aka SPF).
|Temperature-Sensitive||Cdc28ts cells were formed that were wild-type at 25°C but were arrested at G1 at 36°C. It might be that Cdc28 has a high affinity for the wild-type G1 cyclin at 25°C but a low affinity at 36°C.|
|Transformation||Cdc28ts cells were transformed with various plasmids from the wild-type yeast genomic library. Since Cdc28ts had a low affinity for its theorized G1 cyclin at 36°C, then a wild-type phenotype would be restored by massive amounts of the G1 cyclin.|
|High Copy Suppressor||Indeed, a plasmid encoding a cyclin was found to restore a wild-type phenotype to Cdc28ts cells grown at 36°C. Since this plasmid represses the mutant phenotype when present in large quantities, this experiment is known as a high copy suppressor experiment|
What kind of mutation in a mitotic cyclin can lead to entry into mitosis but failure to exit mitosis, i.e. failure to decondense the chromosomes and reassemble the nuclear envelope? MPF (mitotic-cyclin+CDK) drives cells into metaphase but is not needed to continue the cell cycle. In fact, mitotic cyclins must be degraded in order for chromosomes to decondense. Mitotic cyclins all have a conserved sequence at their N-terminus (Arg-X-X-Leu-Gly-X-Ile-Gly-X) that is recognized by APC/C-Cdh1. If this sequence is mutated then mitotic cyclins will not be degraded. This will result in normal mitotic events until the sister chromatids separate in telophase, at which point the sister chromosomes will fail to decondense and the nuclear envelope will not reassemble.