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Identifying substrates of CDK2: cyclin A.

Duncan, Tod; (2001) Identifying substrates of CDK2: cyclin A. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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CDKs and cyclins are the central cell cycle regulators in all organisms studied to date. Full kinase activity of a CDK is only realised upon the binding of a cognate cyclin and phosphorylation on an activating threonine residue. The major cell cycle events, such as entry into S-phase or chromatin condensation, are known to depend on the kinase activity of CDK:cyclin complexes, although the exact changes which these complexes cause to promote particular cell cycle transitions remains unclear. In human cells entry into S-phase is under the control of CDK2 complexed with cyclin E, although soon after entry into S-phase the predominant complex present is CDK2:cyclin A. Two screening methods were developed based on the in vitro expression cloning technique. These screens were employed to identify proteins which could either bind to, or that were substrates of, CDK2:cyclin A. A Xenopus laevis and a HeLa cDNA library were constructed, the cDNA inserts of which could be expressed using in vitro coupled transcription and translation. Pools of plasmid DNA were prepared from the HeLa cDNA library which were used to generate pools of labelled proteins. These pools were then searched for proteins which could bind to micro-affinity columns of CDK2:cyclin A, or which displayed altered electrophoretic mobility through SDS-PAGE in the presence of active CDK2:cyclin A. From these screens, 15 in vitro substrates of CDK2:cyclin A were identified. The localisation of some of these proteins is described. Using these 15 proteins, the substrate specificity of CDK2:cyclin A and CDKl:cyclin B has been examined. The necessity of an intact hydrophobic patch for efficient phosphorylation of substrates has also been investigated and it appears that there is a difference in the way that CDK2:cyclin E and CDK2:cyclin A recognise their substrates.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Identifying substrates of CDK2: cyclin A.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Cell cycle regulators
URI: https://discovery.ucl.ac.uk/id/eprint/10098412
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