[Truncated abstract] The Cdc28 Kinase Subunit (Cks) proteins have an essential role in eukaryotic cell division (Hayles et al., 1986a; Hayles et al., 1986b; Hindley et al., 1987), however their precise function has eluded researchers for over two decades. It is known that Cks proteins bind to and are required for the function of cyclin-dependent kinase (Cdk) proteins during cell cycle progression (Ducommun et al., 1991b; Marcote et al., 1993). p13suc1 (referred to throughout the text as suc1) from the fission yeast Schizosaccharomyces pombe is the archetypal member of the Cks protein family (Hindley et al., 1987). Investigation of Cks proteins within this project is primarily focused on suc1. Two conformations of Cks have been detected crystallographically; a compact monomer in which the C-terminal fourth [beta]-strand inserted into the core of the molecule between strands 2 and 3, and a strand-exchanged dimer in which the fourth [beta]-strand is inserted into the core of the dimer partner in an equivalent position. Cks proteins are classified as "domain swapped" proteins due to their ability to form both compact monomers and strand-exchanged dimers. It is unknown which of these forms occurs in vivo and is biologically relevant. It is likely that the observed strand-exchanged dimeric form of Cks proteins is merely an artefact of the experimental conditions used in crystallographic structure determination. ... The importance of histidine residues in the Cks conformational switch was also demonstrated in investigations of mutants of suc1 in which histidine residues were replaced with alanine. Structural similarity between Cks proteins and a group of proteins known as Peptidyl Prolyl Isomerases (PPIase's) has been recognized. PPIase's catalyse the cis to trans isomerisation of prolyl peptide bonds. The similarity between Cks proteins and the PPIase's prompted investigation of possible enzymatic function of Cks proteins and led to the preliminary demonstration of trans to cis PPIase activity attributable to suc1. An hypothesis has been developed that incorporates the PPIase activity of Cks proteins and a possible mechanism for this function in context with the physical association of Cks proteins with Cyclin-dependent kinases (Cdk's) and the ability of Cks proteins to undergo a conformational switch. According to this hypothesis the substrate molecule of Cdk, with its phosphorylation site in the trans configuration, binds in a hydrophobic cleft on the face of the compact monomeric Cks molecule distal to the Cdk-Cks interface (Bourne et al., 1996). This results in displacement of the ionic or hydrogen bonding interactions of one or more histidine within the Cks molecule by the more basic residues within the substrate motif. This then weakens interactions between the fourth [beta]-strand and the second and third [beta]-strands of the Cks [beta]-sheet allowing the head of the Cks molecule to detach from the C-terminal kinase domain and associate with residues in the N-terminal domain. This simultaneously converts the Ser/Thr-Pro of the substrate to the cis form and places it in or close to the active site of the kinase catalytic subunit facilitating phosphorylation. Given the importance of Cks proteins in regulating cell division processes, insight to be gained through testing this hypothesis will be intrinsically significant to our understanding of eukaryotic cell division.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2007|