1. Calpain 2 Cleavage of Akt in Glioblastoma Multiforme Name: Justin Zhang Mentor: Dr. Jeffrey Greenwood

2. Glioblastoma Malignant brain tumor that is highly invasive Less than 5% of patients survive longer than 5 years. Post-SurgeryPost-Radiotherapy

3. Calpain 2 Is Required For Invasion Is a calcium activated protease Has been shown to be required for invasion of glioma cells Calpain 2 maintains levels of matrix metalloproteinase 2 (MMP2) (2pR) (2pC)

4. Akt Akt is a protein kinase that plays a role in a number of cell functions including apoptosis, cell proliferation and invasion Known to affect levels of MMP2 in glioma cells 3 isoforms of Akt: Akt1, Akt2, Akt3

5. Mechanism of Invasion PIP 2PIP 3 Active Akt PI3K PTEN PDK 1

6. Akt Regulation of Invadopodia Invadopodia are protrusions in the cell membrane that extend into the extracellular matrix Associated with higher levels of MMP2 and invasion There is evidence that Akt is required for invadopodia formation

7. Mechanism of Regulation Hypothesis: Calpain 2 cleavage of Akt regulates Glioblastoma invasion Calpain 2 regulates AktLeads to Invasion

8. Objectives Determine which isoform/s of Akt is being cleaved  Where each isoform of Akt is active in the cell  Determine if the isoform is co-localized with Calpain2 P P C-term N-term PH Domain Catalytic Domain Thr308 Ser473

9. Phosphorylated Akt Localized to Membrane F-Actin Pser473

10. Localization of Akt Isoforms in Glioblastoma Cells Akt1 Akt2 Akt3 F-actin

11. Calpain 2 Localization with F-Actin Calpain 2F-ActinDAPI

12. Akt Isoform Co-Localization with Calpain 2 Akt1Akt2Akt3Calpain2 Nuclear Staining +++++- Perinuclear Staining ++++ Cytosolic Staining +++ + Membrane Ruffle Staining -+++++++ Akt2 and Akt3 localized to same structures as Calpain2 Suggests Calpain2 could be cleaving Isoforms 2 and 3

13. Calpain2 Cleavage of Akt2  Used gel electrophoresis to separate protein samples by molecular weight  Western blotting to identify isoform specificity 72 55 43 34 Blot for Akt2 Control KD Full Length Akt

14. Cortactin Indicates Presence of Invadopodia Invadopodia needed to clear out extracellular matrix to allow for invasion Glioblastoma cells cultured on gelatin and stained for cortactin Invadopodia

15. Invadopodial Staining for Pser473 KD Pser473 KD Cortactin Control Pser473 Control Cortactin

16. Conclusions Calpain2 appears to be cleaving and regulating Akt2 and Akt3 in the invadopodia Calpain2 is required for the turnover of invadopodia Calpain2 Akt2 and 3 ECM

17. Acknowledgements Dr. Jeffrey A. Greenwood Dr. Kevin Ahern Howard Hughs Medical Center (HHMI) URISC Cripps Scholarship Oregon State University