Calpain 2 Cleavage of Akt in Glioblastoma Multiforme Name:
Justin Zhang Mentor: Dr. Jeffrey Greenwood
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Glioblastoma Malignant brain tumor that is highly invasive Less
than 5% of patients survive longer than 5 years.
Post-SurgeryPost-Radiotherapy
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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)
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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
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Mechanism of Invasion PIP 2PIP 3 Active Akt PI3K PTEN PDK
1
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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
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Mechanism of Regulation Hypothesis: Calpain 2 cleavage of Akt
regulates Glioblastoma invasion Calpain 2 regulates AktLeads to
Invasion
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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
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Phosphorylated Akt Localized to Membrane F-Actin Pser473
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Localization of Akt Isoforms in Glioblastoma Cells Akt1 Akt2
Akt3 F-actin
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Calpain 2 Localization with F-Actin Calpain 2F-ActinDAPI
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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
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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
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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
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Invadopodial Staining for Pser473 KD Pser473 KD Cortactin
Control Pser473 Control Cortactin
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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
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Acknowledgements Dr. Jeffrey A. Greenwood Dr. Kevin Ahern
Howard Hughs Medical Center (HHMI) URISC Cripps Scholarship Oregon
State University