1. Visual Snapshots of Intramolecular PKC Activity at the Onset of Mitosis MARM 2008 05/18/2008 Zhaohua Dai Department of Chemistry & Physical Sciences, NY zdai@pace.edu

2. Protein Kinase C Cell proliferation, apoptosis, differentiation, migration Cause cancer, etc. Tools are needed for probing, therapeutics Nakashima, S. J. Biochem. 2002, 132, 669-675.

3. PKC in Early Mitosis (G 2 /M) Review: Black, J. D. Front. Biosci. 2000, 5, 406-423 P. Collas et al J. Cell Sci. 1999, 112, 977-987.

4. PKC  II in G 2 /M Transition A. P. Fields et al. J. Biol. Chem. 1994, 269, 19074-19080. A. P. Fields et al. J. Biol. Chem. 1996, 271, 15045-15053. Target: lamin B Ser 405 85K K m (  M): 4.9 (soluble) and 3.9 (envelope). IC50: 16  M nocodazole Chelerythrine Chelerythrine (PKC  inhibitor ????)

5. NBD-based Fluorescent Sensor for PKC NBD-peptide Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527-11532 Assay PKC  PKC  PKC  Radioact.9.0±1.09.2 ±0.45.0 ±1.0 Fluoresc.29 ±327 ±430 ±5 K m (  M) VIP

6. Caged PKC Sensor Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, 13358-13359 KVIP

7. Why Caged Sensors In cuvette: investigator controls the start and stop of enzyme catalyzed rxns In live cell: the cell controls the timing and during Caged sensors can be delivered in inert forms and activated on demand Give precise temporal control over sensor activity

8. Studying Mitosis Microinjection PtK2 Cells: flat Kangroo rat didney epithelial cells KVIP

9. VIP PKC Activity  Other mitotic kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP) Nek2 (weakly) S. Kumar PKC in PtK2

10. before 0 min injection 2 min uncaging 3 min Green Fl NBD Red Fl 70K dextran- Texas red Coinjection of 200  M KVIP and 5  M 70K dalton texas red-dextran

11. 4 min 5 min 6 min 7 min

12. 0 min injection2 min uncaging25 min Coinjection of 200  M KVIP and 5  M 70K dalton texas red-dextran

13. Injection with 200  M KVIP before NEBD Total cells NEBDLarge enhancement (>40%) Small enhancement (<40%) No enhancement 18Yes 15 690 No 33 1.PKC activity accompanies NEBD. Which one? 2. PKC activity levels off after NEBD: PKC off? or Sensor gone?

14. Injection with 200  M KVIP (Uncaging after NEBD) Total cellsLarge enhancemen t (>40%) Small enhancemen t (<40%) No enhancemen t (within 5%) Very small Enhancement (within 15%) 16 0014 2 1. No PKC activity right after NEBD? 2. Both PKC and phosphatase are active? Right after uncaging Texas red

15. Incubation with 1.5  M okadaic acid No PKC activity right after NEBD. Total cellsNEBDLarge enhancement (>40%)Small enhancement (<40%) No enhancement Little enhancement (around 15%) 10Yes 10 0 082 Phosphatase inhibited

16. High PKC  inhibitor concentration (12  M) induced or blocked cells at prophase 65% of the cells (20 out of 31) are stuck at prophase  II  Nek2 IC 50 1.3  M 11 nM no obs. inhibition Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, 5171-5174 S. Kumar PKC ,  might be implicated in NEBD. Which one?

17. Coinjection w/ 2 mM PKC  inhibitor and 200  M KVIP, 5  M 70K Texas ted-dextran PKC  IC 50 (  M) Ki (  M) 0.0019 0.00080 PKC  385-fold PKC  580-fold PKC  2730-fol PKC  600-fol PKC  1310-fold PKC  1210-fold PKC  940-fold PKC  640-fold Lee, Nandy, Lawrence. JACS, 2004

18. Coinjection of 2 mM PKC  inhibitor and 200  M KVIP Total cellsNEBDLarge enhancement (>30%) Small enhancement (<30%) No enhancement 10Yes 0 000 No 100010 When PKCs are shutdown, NEBD is blocked w/o FL enhancement. Texas-red 30 min after inj

19. Co-injection of 1  M PKC  inhibitor and 200  M KVIP Total cellsNEBDLarge enhancement (>30%) Small enhancement (<30%) No ehancement (within 1%) 15Yes 12651 No 3003 PKC  is responsible for NEBD and FL  1 or  2? PKC  shutdown 14 min after inj Texas-red

20. Redistribution of PKC  I and PKC  II In Cell Cycle N. G. Dulyaninova  1: associated w/ nucleus in interphase and prophase.  2: everywhere in interphase Partial relocation to nuclear boundary in prophase. Significant for NEBD?

21. Conclusion for Caged PKC Sensor Caged sensors can be used to probe PKC activity at G2/M in live cells with temporal precision, providing a way to interrogate enzymatic activity at any point during the cell-division cycle. PKC  is implicated in NEBD of PtK2 cells. It is active just prior to NEBD, not immediately after. Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol. 2007, 14, 1254-1260.

22. Acknowledgement Prof. David S. Lawrence (Einstein, UNC) Dr. Williem Veldhuyzen, Dr. Sandip Nandy Prof. Sanjai Kumar (Einstein, Queens College) Prof. Anne R. Bresnick (Einstein) Dr. Natalya G. Dulyaninova Dr. Zhonghua (Alice) Li NIH (DSL, ARB) Pace University: Startup Fund Scholarly Research Fund