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

2. Outline Background Information:Background Information: –S6K1 –S6K2 –Immune System Review HypothesisHypothesis Materials and MethodsMaterials and Methods ResultsResults Future ExperimentsFuture Experiments

3. S6K1 70kD serine/threonine kinase found in the cytoplasm70kD serine/threonine kinase found in the cytoplasm Phosphorylates S6 on the 40S ribosomal subunitPhosphorylates S6 on the 40S ribosomal subunit Translational UpregulationTranslational Upregulation High levels in growing or proliferating cellsHigh levels in growing or proliferating cells KO mice were smaller, but still able to phosphorylate S6?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.54kD ser/thr kinase homologous to S6K1 in the catalytic and regulatory domains. Predominantly found in the nucleus.Predominantly found in the nucleus. Phosphorylates S6 in vitro.Phosphorylates S6 in vitro. Overlapping and Discrete rolesOverlapping and Discrete roles Immune System: Rapamycin sensitiveImmune System: Rapamycin sensitive

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

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

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

8. Lymphocyte Review B lymphocytes and T lymphocytesB lymphocytes and T lymphocytes B lymphocytes express surface antibodies as well as secrete them.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.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.Activated T lymphocytes synthesize multiple proteins, proliferate and differentiate.

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

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

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

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

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

14. Kinase Assay continued: GST-S6 &  32 P-ATP SDS-PAGE and Coomassie Blue

15. Kinase Assay continued: X-Ray Photography

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.Look for other downstream effectors using S6 phosphorylation as a control. Construct mutant cell lines and observe differences in S6K2 activity: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.Analyze effects of S6 phosphorylation. Look at types and levels of protein synthesis.Look at types and levels of protein synthesis. Complete our understanding of the pathway involved in S6K2 regulation.Complete our understanding of the pathway involved in S6K2 regulation. Analyze other ways of S6K2 activation:Analyze other ways of S6K2 activation: –TCR crosslinking –IL-2 autocrine signaling –Specific signal transduction pathways important in lymphocyte proliferation.

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