REGULATION OF CDK DEPHOSPHORYLATION

Cell cycle progression is orchestrated by the activity of a Cyclin dependent kinase (Cdk) in complex with a Cyclin. The key regulator of early mitotic events is the Cyclin B1/Cdk1 complex. In G2, Cyclin B1/Cdk1 is kept inactive by phosphorylation in the active site of Cdk1. In humans, three members of the Cdc25 family of dual-specificity phosphatases, Cdc25A, Cdc25B and Cdc25C activate Cyclin/Cdk complexes. We sought to better understand the regulation of Cyclin B1/Cdk1 activation in mitotic entry. A working hypothesis has been that the cell partly controls Cyclin B1/Cdk1 activation by regulating the localisation of Cdc25 proteins. Therefore, we have studied the intracellular localisation of Cdc25A and Cdc25B. We find that both Cdc25A and Cdc25B are predominantly nuclear, although they continuously shuttle between the nucleus and the cytoplasm. We identify nuclear export sequences (NES:s) in both Cdc25A and Cdc25B. Although the Cdc25A NES and the Cdc25B NES are very similar, only the latter is sensitive to an inhibitor of Exportin-1. We map a nuclear localisation signal (NLS) in Cdc25A, and confirm a previously reported NLS in Cdc25B. In response to UV-light, Cdc25B partly translocates to the cytoplasm. The translocation is dependent on the Cdc25B NES, p38 MAPK activity and the integrity of a 14-3-3 binding site. We propose that in response to stress, p38 MAPK mediates 14-3-3 binding to Cdc25B, which results in its cytoplasmic sequestration. We also investigated the roles of the individual Cdc25s by reducing their levels with RNA interference (RNAi). Cells treated with siRNA to Cdc25A or to Cdc25B, but not to Cdc25C, were delayed in mitotic entry. Moreover, cells treated with siRNA to both Cdc25A and Cdc25B were blocked in G2, suggesting that Cdc25A and Cdc25B co-operate to induce mitosis. Data from time-lapse movies suggest that whereas Cdc25A may mainly regulate nuclear events, Cdc25B probably regulates events in both the nucleus and the cytoplasm. Cells treated with siRNA to Cdc25B, but not to Cdc25A, contain higher levels of phosphorylated Cyclin B1/Cdk1 on unseparated centrosomes than control cells. This shows that Cdc25B is involved in the process leading to the accumulation of active Cyclin B1/Cdk1 on the centrosomes. By following the Cyclin B1/Cdk1 activation with phospho-specific antibodies, we note that active Cyclin B1/Cdk1 first appears on the centrosomes, shortly before they start to migrate apart. The activation then spreads to the cytoplasm, and when approximately 50% of the complexes are active, Cyclin B1/Cdk1 translocates to the nucleus. During translocation, the Cyclin B1/Cdk1 activation proceeds similarly in the nucleus and the cytoplasm. We do not see an inactivation of Cyclin B1/Cdk1 in late metaphase. Our data are in support of bistability governing Cyclin B1/ Cdk1 activation. This thesis is based on the following articles and manuscripts, which will be referred to by their Roman numerals: I. Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress. Arne Lindqvist, Helena Kallstrom and Christina Karlsson Rosenthal. Journal of Cell Science. 2004. 117: 4979-4990 II. Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import. Helena Kallstrom, Arne Lindqvist, Vitek Pospisil, Andreas Lundgren and Christina Karlsson Rosenthal. Experimental Cell Research. 2005. 303: 89–100 III. Cdc25B co-operates with Cdc25A to induce mitosis but has a unique role in activating Cyclin B1/Cdk1 at the centrosome. Arne Lindqvist, Helena Kallstrom, Andreas Lundgren, Emad Barsoum and Christina Karlsson Rosenthal. Submitted. IV. Gradual activation of the Cdk1 pool in mitosis. Arne Lindqvist and Christina Karlsson Rosenthal. Manuscript. 1. Wordlist and abbreviations a Short wordlist for non-biologists 1 b Abbreviations 2 2 Introduction a The eukaryotic cell cycle 3 1 History 2 Basic mechanisms 3 Mitotic entry 4 Cyclin/Cdk complexes in mitotic entry b Regulation of Cyclin/Cdk activity in mitotic entry 6 1 Transcription 2 Proteolysis 3 Inhibitors 4 T-loop phosphorylation 5 Localisation 6 Active site phosphorylation 7 Models of Cdk1 activation c Cdc25 phosphatases 8 1 In different species 2 Cdc25 and 14-3-3 3 Localisation 4 In the absence of Cdc25 5 Cdc25 and Cancer d The cell cycle in stress 11 1 The concept of checkpoints 2 ATM/ATR 4 p38 MAPK 3 Objectives and Methods a Objectives and aims 13 b Methodological considerations 15 1 Localisation 2 Quantification of fluorescence 3 RNA interference 4 Using transformed cell-lines 5 Synchronisation 4 Results and discussion a Cdc25B localisation (I) 18 b Cdc25A localisation (II) 19 c Cdc25 function (III) 20 d Cdk1 activation (IV) 21 CONTENTS