Activation of S6K2 in lymphocytes by PMA+Ionomycin.

1. Activation of S6K2 in lymphocytes by PMA+Ionomycin. By Bonnie Larsen

2. Outline • Background Information: • S6K1 • S6K2 • Immune System Review • Hypothesis • Materials and Methods • Results • Future Experiments

3. S6K1 • 70kD serine/threonine kinase found in the cytoplasm • Phosphorylates S6 on the 40S ribosomal subunit • Translational Upregulation • High levels in growing or proliferating cells • KO mice were smaller, but still able to phosphorylate S6?

4. S6K2 • 54kD ser/thr kinase homologous to S6K1 in the catalytic and regulatory domains. • Predominantly found in the nucleus. • Phosphorylates S6 in vitro. • Overlapping and Discrete roles • Immune System: Rapamycin sensitive

5. What role does S6K2 play in the immune system? AND How can we study S6K2 in leukocytes?

6. S6K1 in lymphocytes • S6K1 levels high in T-cell lines. • Sensitive to Rapamycin and Wortmannin • CD-5, CD-28, IL-2, TCR crosslinking, PMA+Ionomycin (P+I) • B-cell surface receptor ligation

7. S6K2 in lymphocytes • Expressed at high levels in leukocytes and spleen • Regulated by PI3K and mTOR • ??

8. Lymphocyte Review • B lymphocytes and T lymphocytes • B lymphocytes express surface antibodies as well as secrete them. • T lymphocytes express a T-cell Receptor (TCR) complex and are activated by antigen presenting cells and TCR crosslinking. • Activated T lymphocytes synthesize multiple proteins, proliferate and differentiate.

9. T-cell signaling Co-receptor TCR Crosslinking IL-2 Autocrine Signaling Proliferation Protein Synthesis Differentiation

10. How can we visualize S6K2 activation in T lymphocytes? • PMA+Ionomycin mechanism of action • By-pass TCR crosslinking • If S6K2 activation occurs, it will phosphorylate S6. • We can measure changes in S6 phosphorylation to analyze S6K2 activity.

11. Activation: PMA Ionomycin PMA+Ionomycin Time Coursed Assay S6K2 vs. S6K1 Rapamycin Sensitivity: 0,5,10,15,20,25,30,60 Activation 20minutes incubation minutes by P+I with Rapamycin prior Activation with P+I to P+I Activation Experimental Design Kinase Assay Phosphorimaging

12. Cell Lysis J77/U937 P I Experimental Design-Kinase Assay

13. Kinase Assay continued… Anti-S6K1/S6K2 PAS Beads Wash

14. GST-S6 & g32P-ATP SDS-PAGE and Coomassie Blue Kinase Assay continued:

15. X-Ray Photography Kinase Assay continued:

16. Expected Results AND What they can tell us…

17. PMA+Ionomycin activates S6K2 but not PMA or Ionomycin alone.

18. 20-30min. activation time with P+I yields significant S6K2 activity.

19. P+I activation of S6K1 vs. S6K2

20. P+I activation of S6K2 is Rapamycin Sensitive.

21. Simplified overview of S6K2 regulation in lymphocytes:

22. What knowledge has been gained? Where do we go from here?

23. Now that we know what to expect with S6 phosphorylation by S6K2, we can… • Look for other downstream effectors using S6 phosphorylation as a control. • Construct mutant cell lines and observe differences in S6K2 activity: • Can S6K2 still be activated for phosphorylation if we disrupt certain other cellular factors? • If we change certain amino acids in the primary structure of S6K2 is activity increased or decreased?

24. Future Experiments Continued… • Analyze effects of S6 phosphorylation. • Look at types and levels of protein synthesis. • Complete our understanding of the pathway involved in S6K2 regulation. • Analyze other ways of S6K2 activation: • TCR crosslinking • IL-2 autocrine signaling • Specific signal transduction pathways important in lymphocyte proliferation.

25. Acknowledgements • Dr. Lee-Fruman • Deborah, Sharsti, Sopheap, Hemant and Derek • Howard Hughes Medical Research Institute