Mitosis: Cyclin-CDKs

By Levi Clancy for Student Reader on
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cell with wild-type low and high levels of mitoti cdk

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:

CyclinsThe 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.
InhibitorsS-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.
DegradationMitotic-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.
Cyclin-CDKNick-NameOverview
Mitotic-Cyclin+CDKMPFTriggers the formation of mitotic spindle. Promotes mitosis i.e. chromatin condensation. Causes nuclear envelope breakdown by phosphorylating the lamins that form an intermediate filament-type network (nuclear lamina) underlying the inner nuclear membrane. The three lamins present in the nuclear lamina, lamin A,B & C, are phosphorylated by MPF at serine amino residues. This leads to depolymerisation of nuclear lamina & breakdown of nuclear envelope into small vesicles.
VertebrateS. cerevisaeS. pombeRoleOverview
Cyclin BCdc28Cdc13Mitotic CyclinMitotic cyclins must be degraded by APC/C-Cdh2 for chromosomes to decondense; however, this degradation is not needed for sister chromatids to separate during anaphase. 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-Cdh2; without this sequence, mitotic cyclins are not degraded. Cyclin-B+CDK1 is MPF in Xenopus and S. pombe.
CDK1Cdc28Cdc2Mitotic CDKMPF is Cyclin-B+CDK1 in Xenopus and S. pombe.
repression and activation of mitosis promoting factor (maturation promoting factor) MPF
Cyclin-CDKNick-NameOverview
Mid-G1-cyclin+CDKSPFKnown as the s-phase promoting factor, as it is required to initiate DNA synthesis. Activates transcription factors for genes encoding the S phase cyclins and Sic1, and for genes needed for dNTP and DNA synthesis. Also, the G1 cyclin-CDK inactivates Cdh2, which would have destructed the S phase and mitotis cyclins.
VertebrateS. cerevisaeS. pombeRoleOverview
Cyclin DCln3Cdc13Mid G1 CyclinTranscription "growth" factors activating Cyclin D and CDK4/6 are encoded by delayed response genes.
CDK4 & CDK6Mid G1 CDKTranscription "growth" factors activating Cyclin D and CDK4/6 are encoded by delayed response genes.
cell cycle mitosis biochemical pathway cyclin cdks
Human CDKs and corresponding Cyclins
CDK1cyclin A, cyclin BCDK2cyclin A, cyclin ECDK3CDK4cyclin D1, D2, D3
CDK5p35 (a regulator dissimilar to cyclins)CDK6cyclin D1, D2, D3CDK7cyclin HCDK8cyclin C
CDK9cyclin T1, T2a, T2b, cyclin KCDK10CDK11cyclin 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.

S. pombe

PhaseCyclinCDK
MitosisCdc13Cdc2
S. cerevisiae

PhaseCyclinCDK
Mid G1Cln3Cdc28
Late G1Cln1 & Cln2Cdc28
Early S-PhaseClb5 & Clb6Cdc28
Late S-Phase + Early mitosisClb3 & Clb4Cdc28
Late MitosisClb1 & Clb2Cdc28
Vertebrates

PhaseCyclinCDK
Mid-G1Cyclin D'sCDK4 & CDK6
Late-G1Cyclin ECDK2
S-phaseCyclin ACDK2
MitotisCyclin A &
Cyclin B
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-SensitiveCdc28ts 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.
TransformationCdc28ts 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 SuppressorIndeed, 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

Mutations

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.