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Assays for Cyclin-Dependent Kinase Inhibitors

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Cyclin-dependent kinases (CDK) are serine/threonine kinases that regulate cell cycle control and progression (1 ,2 ). A CDK holoenzyme complex is active if associated with its cyclin partner and if the complex is phosphorylated at specific activating residues (threonine 160/161) (1 ,2 ). The progression through the cell cycle is mediated by the sequential activation of CDKs during different phases of the cycle. The G1/S phase transition of the cell cycle is mediated by phosphorylation of the Retinoblastoma gene product (Rb) by CDK4 and/or CDK6 (G1/S checkpoint), leading to the release of bound E2F from Rb and allowing the transcription of genes by “free E2F” necessary for S phase progression. During late G1 phase, CDK2/cyclin E complex phosphorylates several substrates including Rb. On the other hand, CDK2/cyclin A complex is active throughout S phase. For cells to progress to mitosis, CDK1 (CDC2) must be “activated,” and activated cdc2 phosphorylates substrates required for mitosis (3 ).
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