The cell cycle is one of the fundamental processes in nature, and is primarily concerned with the faithful replication of cellular contents, followed by even division to produce two identical daughter cells. It is made up of five discrete biochemical steps, comprising the interphase (G1, S and G2) and the mitotic phase (mitosis and cytokinesis), with two major regulatory checkpoints at G1 and G2. The focus of this research is the G2 checkpoint, which ensures the successful achievement of DNA replication, prior to the initiation of mitosis. Arrest or progression is principally mediated by the CDK1/cyclin B1 complex; phosphorylation of CDK1 by wee1 kinase prevents progression to mitosis, and subsequent dephosphorylation by the CDC25 phosphatases, initially the B isoform, leads to mitotic onset. The aim of this research was the biophysical and/or biochemical characterisation of the molecular complexes that form at the G2 checkpoint to regulate entry into mitosis. CDK1 and cyclin B1 were separately expressed and purified from baculovirus-infected Sf9 cells. The wee1/14-3-3β complex was also expressed and purified, incorporating either full length wee1 or a truncated version from which the N-terminal domain of wee1 was deleted. Both exhibited wee1 kinase activity, at equivalent levels, p<0.001, with a 2.4 fold increase in kinase activity when wee1 is bound by 14-3-3β, p>0.001. Tryptic digestion of the complex indicated that its architecture was likely to be flexible and open, particularly within the N-terminal domain of wee1. CD analyses indicated that the wee1/14-3-3β complex was folded, with 30-40% α-helical content and 10-20% β-sheet content. Dissociation experiments were unsuccessful, however, indicating a high strength of interaction between wee1 and 14-3-3β. The empirical stoichiometry of the complex was determined as 1:1; subsequent native molecular weight determination suggested that the minimal functional unit is likely to be a 2:2 wee1/14-3-3β arrangement. It was proposed that the structural architecture of this complex may be similar to the serotonin N-acetyltransferase/14-3-3ζ complex. Experiments to determine the structure experimentally, using either TEM or x-ray crystallography, were unsuccessful, as the complex appeared to exhibit a high degree of flexibility in solution. CDC25B was also expressed and purified, and was found to co-purify with a putative Sf9 14-3-3 protein. Consequently, it was re-cloned to co-express with 14-3-3β, and subsequent analysis of the resulting CDC25B/14-3-3β complex indicated that the empirical stoichiometry was 1:1, with the functional organization likely to be a 2:2 arrangement. It was proposed that the structural arrangement of this complex is most likely to be similar to that of the wee1/14-3-3β complex.
| Identifer | oai:union.ndltd.org:ADTP/247891 |
| Date | January 2009 |
| Creators | Collings, Melanie |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Whole document restricted. Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. | Whole document restricted but available by request. Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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