G,/S-Cdk S-Cdk Structure and function of CDKs and Ubiquitin ligases Cyclin-dependent kinases . All kinases have a pocket that holds ATP and an active site that holds the polypeptide chain that will receive the phosphate group · Cyclin-dependent kinases must be bound to a cyclin (protein) to be active. · Cyclins do not have intrinsic enzymatic activity Cyclins cycle but CDK levels are constant · Cyclins undergo a cycle of synthesis and degradation in each cell cycle o Their concentration in a cell is always fluctuating up and down because its synthesis is regulated o We start making mRNA and then accumulating proteins, at which point we destroy the proteins so conc of Cdk goes back down. · In simple cells, Cdk levels are constant (yeast use a single Cdk; vertebrates use 5 Cdks) · Cyclical changes in cyclins drive changes in Cdk-cyclin activity (note- s-Cdk denotes Cdk bound to S cyclins etc.) G,/S-cyclin S-cyclin M-cyclin start G1 G2/M · G1/S-cyclins bind Cdks in G1 and commit the cell to DNA replication o Some cells also require G1 cyclins to pass the restriction point/start point in late G1 · S-cyclins bind Cdks during the S phase and are required to initiate DNA replication . M-cyclins promote the events of mitosis Cdks also require phosphorylation of the T-loop · Incredibly important otherwise cell will die . Cyclin binds to the Cdk . This will change the shape of the protein around the active site · T-loop becomes more accessible (sticks out of the kinase) · Cdk-activating kinase (CAK) will phosphorylate the T-loop so it holds its position in an open conformation (The first layer of regulation is the cyclin. Only once the cyclin is bound can the CAK phosphorylate the T-loop) The T-loop · The T-loop is a "pseudo-substrate . It inhibits the activity of the Cdk by partially occupying the active site · When CAK phosphorylates the T-loop, the position of the T-loop shifts and the Cdk is able to bind to the substrate · Since CAK is always active, phosphorylation of the T-loop is not a limiting step although it is required. · metaphase-anaphase S G2 M „cyclin APC/C M-Cdk Regulation of cyclins . The transcription of cyclin mRNA controls when cyclins appear in the cell cycle . Degrading cyclins is a way of ensuring that the cell cycle does not go backwards · All remaining cyclins are degraded upon entry into anaphase, so levels are very low at the start of G1. Cyclins influence the target specificity of the Cdk G1 Fine-tuning the activity of Cdk-cyclins . The activity of the Cdk-cyclin complex is fine-tuned by the balance between inhibitory kinases (kinases that inhibit by adding Pi) and activating phosphatases (kinases that activate by removing Pi) · CKIs (CDK-inhibitors) are another way to reversibly inhibit CDK-cyclin complexes.
The balance between Wee1 and Cdc25 determines Cdk-cyclin activation · Wee1 kinase (inhibitory kinase) adds a phosphate to Cdk, inhibiting it. · Cdcfi5 (phosphatase) takes off the inhibitory phosphate, activating the