Laboratory of Molecular Biophysics Laboratory Journal 2000 Dr. J. A. Endicott

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Jane A. Endicott

Cell Cycle Proteins.

Introduction

Sequential activation of members of the cyclin-dependent protein kinase family (CDKs) orders the events required for DNA replication and cell division. Both the CDK and cyclin families have multiple members, but only CDKs 1, 2, 4 and 6, when bound to their cognate cyclins, appear to have major roles in controlling cell cycle progression (reviewed in [1]). These complexes are either pivotal to the integration of multiple signal transduction pathways into the cell cycle or they are the downstream targets of cell cycle checkpoint pathways. They are tightly regulated through mechanisms that include inhibitory phosphorylation, association with additional regulatory molecules, and subcellular localisation. This year we have continued our studies on the structural consequences of CDK phosphorylation by determining structures for CDK2/cyclin A complexes phosphorylated on Tyr15 and Thr160 in the presence and absence of Mg-AMPPNP. A second research area is in the characterisation of interactions between CDKs and their regulatory molecules. This year we have focussed on working towards a molecular description of the structure and function of the SKP1/SKP2/CDK2/cyclin A and CDK2/cyclin A/Cks1 complexes.

An increasingly important mode of cell cycle control appears to be the targeted ubiquitination of cell cycle regulatory proteins, their directed degradation often being responsible for triggering key events in cell cycle progression. [2], [3]. The pathway consists of three enzymes/enzyme complexes. In an ATP-dependent first step, the ubiquitin-activating protein (E1) activates ubiquitin that is then transferred to the active site cysteine of an ubiquitin-conjugating enzyme (E2). E2s can directly transfer the ubiquitin to the target protein but many act in conjunction with a third enzyme, an ubiquitin ligase (E3) which plays a role in substrate selection. E3s are diverse and are frequently multi-protein complexes. At least three major cell cycle transitions require specific degradation of regulatory proteins: entry into S-phase, separation of sister chromatids and exit from mitosis. Two types of E3 that regulate cell cycle progression have been well characterised: SKP1, cullin, F-box complexes (SCFs) responsible for regulatory events at the G1-S transition [3], and the anaphase promoting complex (APC) that acts at the end of mitosis and controls passage back into the G1 phase of the cell cycle [4]. This year we have continued structural and functional characterisation of the SCF^SKP2 and APC E3 ligase complexes

Aberrant CDK activity is a common defect in a variety of human tumours. We continue to have an interest in the determination of structures of CDK2 and CDK2/cyclin A in complex with various small molecules with the aim of designing potent and selective CDK inhibitors.

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