1. A novel DAG-dependent mechanism links PKC  and Cyclin B1 regulating the G2/M progression of cell cycle Alessandro Poli, Giulia Ramazzotti, Alessandro Matteucci, Ester Orsini, Gabriella Giuliani-Piccari and Matilde Y. Follo Cellular Signalling Laboratory (Head L. Cocco) Department of Biomedical Sciences, University of Bologna Membrane, Morphology and Function, 5-8 May 2014, Fara San Martino, Italy

2. Protein kinases C and cell cycle

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4. PKC  affects Cyclin B1 levels in K562 cell line Block in G2/M using Nocodazole for 16h at 20ng/ml * 3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione anilinomonoindolylmaleimide ** Phorbol myristate acetate * G2/M 90% **

5. Ctrl Scrambled PKC  KD PKC inhibitor

6. * PKC  DN ---- Lys 376 in Arg * WT DN Empty

7. Lack of PKC  drives to a higher Cyclin B1 degradation

8. Co-localization and interaction of PKC  and Cyclin B1 during different cell cycle phases HBSS o/n RPMI 10% FBSNocodazole

9. PKC  involvement in Cyclin B1 nuclear accumulation at G2/M checkpoint Nocodazole 16 h 20ng/ul

10. Nocodazole 6 h 20ng/ul

11. Nuclear DAG accumulation during G2/M progression is necessary for PKC  and Cyclin B1 translocation from cytoplasm

13. Nuclear PLC  1 is responsible for DAG fluctuations during cell cycle progression PLC  1 KD Scrambled Radioactivity (c.p.s.) Time (minutes) IP3 PLC assay Radioactivity (c.p.s) PLC  1 KD Scrambled Nuclei

14. Novel mechanism for G2/M regulation in K562 cells PKC  Cyclin B1 PKC  Cyclin B1 PLC  1 PIP2 DAG p21 Ub Cyclin D3 Lamin B1 P Cdk1 G2/M progression Cyclin B1 degradation Cytoplasm G2/M checkpoint Nucleus

15. Aknowledgements Laboratory Director Prof. Lucio Cocco Staff Sara Mongiorgi Matilde Y. Follo Giulia Ramazzotti Alessandro Matteucci Annalisa Lonetti Roberta Fiume Irene Faenza THANK YOU FOR YOUR ATTENTION

16. Thank you

17. Co-localization and interaction of PKC  and Cyclin B1 during different cell cycle phases HBSS o/n RPMI 10% FBSNocodazole

19. PKC   -tubulin cdk7 P-cdc2 tyr 15 cdc25c cdc2 Lamin A/C Cyclin B1  -tubulin Cyclin B1 P133 Cyclin B1 P147