The eukaryotic cell cycle is driven by a set of kinases called the cyclin-dependent kinases (CDKs). CDK2 in complex with cyclin A is involved in progression through late G1 and S phase. During late G1, CDK2/cyclin A phosphorylates the CDK inhibitor p27 leading to its ubiquitinylation by the SCFSkp2 complex and subsequent degradation. Skp2 is the substrate recognition component of this complex and has been found to be up-regulated in many cancers and high levels 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 recruitment of p27 and potentially other substrates. Skp2 is itself degraded by the APCCdh1 E3 ubiquitin ligase complex via an interaction that is modulated by post-translation modifications to the Skp2 N-terminal sequence prior to the start of the F-box. This sequence includes a CDK2 phosphorylation site and it is not known what role CDK2/cyclin A phosphorylation and association play in the regulation of Skp2 activity and stability.

The aims of this project are 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 has been used to identify the regions on cyclin A and Skp2 which are mediating this interaction. The ability of these mutants to form a complex has been evaluated by size-exclusion chromatography and isothermal titration calorimetry (ITC). Residues on a loop region of cyclin A were identified as crucial to the Skp2 interaction. Mutations in this region do not interfere with binding to CDK2, p27 or p107. They also do not affect CDK2/cyclin A kinase activity towards pRb and histone H1. CDK2 was found not to have a role in binding to Skp2 as Skp2 binds to cyclin A and to the CDK2/cyclin A complex with similar affinity.

Cyclin A was found to bind to an extended region within the N-terminus of Skp2. Residues L32, L33, S39 and L41 were confirmed as being part of this cyclin A binding region (Ji, P., et al. 2006). The Skp2 N-terminal region is involved in binding many regulatory proteins such as 14-3-3β and undergoes extensive post-translational modification. The binding of cyclin A to this sequence may affect subsequent Skp2 post-translational modification through affecting its structure and/or accessibility to modifying enzymes. There is a requirement for Skp2-cyclin A interaction during apoptosis, and for Skp2 degradation and p27 regulation. The functional significance of the Skp2-cyclin A interaction described here is currently being characterized through cell-based assays.

Ji, P., et al. (2006). “Skp2 contains a novel cyclin A binding domain that directly protects cyclin A from inhibition by p27Kip1.” J Biol Chem 281(33): 24058-24069.

Citation Format: Bailey C. Massa, Mathew P. Martin, Martin E. Noble, Jane A. Endicott. Structural and functional characterization of Skp2-containing complexes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2015-3767