Structural and functional characterisation of Skp2-containing complexes

Abstract

The eukaryotic cell cycle is driven by a set of kinases called the cyclin-dependent kinases (CDKs). During G1, CDK2/cyclin E phosphorylates the CDK inhibitor, p27, leading to its ubiquitination by the SCFSkp2 complex and subsequent degradation. Skp2 is the substrate recognition component of the SCFSkp2 complex and has been found to be up-regulated in many cancers. High levels of Skp2 indicate poor prognosis. Its overexpression often correlates with low p27 levels. Skp2 was first identified in a complex with CDK2, cyclin A, Cks1 and Skp1. CDK2/cyclin A binds to SCFSkp2 and is required for the recruitment of p27 to the SCFSkp2 complex. The aims of this project were to structurally characterize the CDK2/cyclin A/Skp1/Skp2 complex and to identify the functional significance of the Skp2/cyclin A interaction. In the absence of a crystal structure, site-directed mutagenesis was used to identify the regions on cyclin A and Skp2 which mediate this interaction. Mutation of residues 244-247 of cyclin A blocked its interaction with Skp2. Mutation of this region did not interfere with binding to CDK2, or p27, and CDK2/cyclin A kinase activity did not appear to be affected. CDK2 was found not to have a role in binding to Skp2. Cyclin A binds to an extended region within the N-terminus of Skp2. Residues Leu32, Leu33, Ser39 and Leu41 of Skp2 have been reported to comprise the cyclin A binding region (Ji et al., 2006). This site was confirmed and a further two residues were identified as contributing to the interaction. The Skp2 N-terminal region is involved in binding many regulatory proteins and is heavily post-translationally modified. The binding of cyclin A to this sequence may therefore affect the structure and/or accessibility to modifying enzymes. The characterisation of these mutants has shown that they are potentially useful tool mutants for studying Skp2 function and regulation in human cell-based assays.