1. Calpain 2 regulation of Akt in glioblastoma cell invasion
Presented by Maria Nguyen
Guidance of Dr. Jeffrey A. Greenwood

2. Glioblastoma is highly invasive
Less than 5% chance of survival over 5 year span
Pre-surgery
Post-surgery
12 months
17 months
Glioblastoma
Nakada et al Cell. Mol. Life Sci. 64:

3. Unique mutation in glioma cells
PIP2 X
PTEN
PTEN
PI3-K
PIP3
Akt P
Akt
Localizing it to the plasma membrane.
INVASION

4. pAkt localized to membrane projections
F-Actin
Alexa 555
100x imaging with controls for bleed through. Can see that Akt is localized to the membrane ruffles and not on the Filamentous Actin. Where protrusions start forming for cell migration.
In the process of singling out a specific phosphatase of interest and immunostaining for that protein. If they are localized in the same region it would suggest that the phosphatase was involved as a turnover mechanism for Akt phosphorylation.
Fixed/Extract.

5. Unique autocrine glutamate system
Ca2+
Ca2+
Ca2+
Glutamate
Calpain 2
In human glioma cells, large fluxes of Calcium constant cycle within the cells.
Ca2+
INVASION
Ca2+
Ca2+

6. Calpain 2 is required for invasion
Decreased by
90%
Matrigel coated transwells with or without 10% FBS-DMEM (serum) as a chemoattractant in the lower wells.
Incubation for three days.
Jang et al Neurochemical Res.

7. Calpain 2 maintains MMP2 levels
The Greenwood lab used shRNA along with an antibiotic to knockdown Calpain 2 in U87MG cells .
Calpain 2 required for invasion through regulation of extracellular MMP2 concentrations.
Actin
Matrix metalloproteinase 2
(MMP2)
Yamaguchi et al Curr. Op. Cell Biol. 17:

8. What are the mechanisms of regulation?
Calpain 2
MMP2 and Akt:
Akt involved in the expression of MMP2
Either…
Directly
Indirectly ?
Akt
MMP2
Invasion

9. Testing pAkt levels pAkt (Ser-473) Actin 72kDa 55kDa 43kDa Control KD
Do I need to explain what a Western blot is?

10. Time dependent dephosphorylation
LY294002 SD 0’ 5’
15’
30’
60’
70kDa
55kDa
Control
70kDa KD
55kDa
PIP2 X
PI3-K
Describe the LY procedure.
PIP3 X
Akt P
Akt

11. Calpain 2 regulates proteolysis of Akt
Control KD
100kDa
Calpain 2
70kDa
70kDa
pAkt (Ser-473)
55kDa
25kDa
BDPs

12. Calpain 2 Calpain 2 Working Hypothesis
Calpain 2 directly cleaves Akt in the regulation of glioblastoma cell invasion.
Calpain 2
Ca2+
Calpain 2
Ca2+
Originally: Thought that a phosphatase was regulating the levels of pAkt
Now: Calpain 2 directly cleaves Akt.

13. Akt unfolds and is susceptible to cleavage
Add PIP3 on the PH domain
PIP3 P P
N-term
C-term

14. Proposed site of cleavage
Calpain 2
Ca2+ PH
domain
Catalytic
domain
Regulatory
domain
N-term
Catalytic
domain PH
Thr 308
Ser 473
PIP3 P
C-term
N-term
Calpain 2 cleavage of Akt could mean:
Inactivation
Phosphorylation independent activation
About 296
Site of cleavage for a 22 kDa fragment is estimated to lie in the middle of the catalytic domain

15. Significance
I propose calpain 2 regulates Akt activity through cleavage:
Identified a novel mechanism for the regulation of invasion.
Pre-surgery
Post-surgery
12 months
17 months
Glioblastoma
Nakada et al Cell. Mol. Life Sci. 64:

16. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern
Dr. Jeffrey Greenwood
Greenwood Laboratory Members:
Sangeet Lal, M.S.
OSU Biochemistry and Biophysics Department