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Calpain 2 Cleavage of Akt in Glioblastoma Multiforme

Calpain 2 Cleavage of Akt in Glioblastoma Multiforme. Name: Justin Zhang Mentor: Dr. Jeffrey Greenwood. Glioblastoma . M alignant brain tumor that is highly invasive Less than 5% of patients survive longer than 5 years. Post-Surgery. Post-Radiotherapy. Calpain 2 Is Required For Invasion.

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Calpain 2 Cleavage of Akt in Glioblastoma Multiforme

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  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-Surgery Post-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 maintainslevels of matrix metalloproteinase 2 (MMP2) (2pC) (2pR)

  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 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 regulates Invasion Akt Leads to Calpain 2

  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 Catalytic Domain PH Domain P P Thr308 Ser473 C-term N-term

  9. Phosphorylated Akt Localized to Membrane Pser473 F-Actin

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

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

  12. Akt Isoform Co-Localization with Calpain 2 • 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 Blot for Akt2 KD Control 72 Full Length Akt 55 43 34

  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. InvadopodialStaining for Pser473 Control Pser473 KD Pser473 KD Cortactin 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 ECM Akt2 and 3

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

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