Calpain 2 Cleavage of Akt in
Glioblastoma Multiforme
Name: Justin Zhang
Mentor: Dr. Jeffrey Greenwood
Glioblastoma
 Malignant 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
 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)
(2pC)
(2pR)
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
Mechanism of Invasion
PIP 2
PI3K
PTEN
PIP 3
PDK 1
Active Akt
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
Mechanism of Regulation
 Hypothesis: Calpain 2 cleavage of Akt regulates
Glioblastoma invasion
Calpain 2
regulates
Akt
Leads to
Invasion
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
PH
Domain
N-term
Catalytic Domain
Thr308
P
Ser473
P
C-term
Phosphorylated Akt Localized to Membrane
F-Actin
Pser473
Localization of Akt Isoforms in Glioblastoma Cells
Akt1
F-actin
Akt2
F-actin
Akt3
F-actin
Calpain 2 Localization with F-Actin
Calpain 2
F-Actin
DAPI
Akt Isoform Co-Localization with Calpain 2
Akt1
Akt2
Akt3
Calpain2
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
Calpain2 Cleavage of Akt2


Used gel electrophoresis to separate protein samples by
molecular weight
Western blotting to identify isoform specificity
Blot for Akt2
Control
KD
72
Full Length Akt
55
43
34
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
Invadopodial Staining for Pser473
Control
Pser473
Control
Cortactin
KD Pser473
KD Cortactin
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
Acknowledgements
 Dr. Jeffrey A. Greenwood
 Dr. Kevin Ahern
 Howard Hughs Medical Center (HHMI)
 URISC
 Cripps Scholarship
 Oregon State University