CALPAIN 2 REGULATION OF AKT IN
GLIOBLASTOMA CELL INVASION
Presented by Maria Nguyen
Guidance of Dr. Jeffrey A. Greenwood
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. 2007. Cell. Mol. Life Sci. 64: 458-478.
Unique mutation in glioma cells
X
PIP2
PTEN
PI3-K
PIP3
Akt
INVASION
P
Akt
pAkt localized to membrane projections
F-Actin
Alexa 555
Unique autocrine glutamate system
2+
Ca2+
2+
Ca2+
2+
Ca2+
Glutamate
Calpain 2
2+
Ca2+
INVASION
2+
Ca2+
2+
Ca2+
Calpain 2 is required for invasion
Decreased by
90%
Jang et al. 2010. Neurochemical Res.
Calpain 2 maintains MMP2 levels
Actin
Matrix metalloproteinase 2
(MMP2)
Yamaguchi et al. 2005. Curr. Op. Cell Biol. 17: 559-564.
What are the mechanisms of regulation?
Calpain 2
Either…
Akt
MMP2 and Akt:
1. Directly

?
 Akt
MMP2
Invasion
involved in the expression
2. Indirectly
of MMP2
Testing pAkt levels
72kDa
pAkt (Ser-473)
55kDa
Actin
43kDa
Control
KD
pAkt /Actin
1.5
1
Control
KD
0.5
0
UG87MG cell lines
Time dependent dephosphorylation
LY294002
70kDa
SD
0’
5’
15’
30’
60’
Control
55kDa
70kDa
KD
55kDa
100
% of control
PIP280
60
X
PI3-K
40
PIP320
Akt
0
X
Control
KD
P
0
20
40
Time
Akt
60
Calpain 2 regulates proteolysis of Akt
100kDa
70kDa
70kDa
55kDa
25kDa
50
KD
Calpain
2
40
% of total Akt
Control
30
pAkt (Ser-473)
20
10
BDPs
0
U87MG cell lines
Control
KD
Working Hypothesis
Calpain 2 directly cleaves Akt in the regulation of
glioblastoma cell invasion.
2+
Ca2+
2+
Ca2+
Calpain
2
Calpain 2
Akt unfolds and is susceptible to
cleavage
PIP3
N-term
P
P
C-term
Proposed site of cleavage
PH
PH Catalytic
domain domaindomain
Catalytic
domain
Thr 308
P
PIP3
Regulatory
domain
Ser 473
P
N-term
N-term
C-term
Aboutmean:
296
Calpain 2 cleavage of Akt could
Site 1.
of Inactivation
cleavage for a 22 kDa fragment is estimated to
in the middle of
the catalyticactivation
domain
2. lie
Phosphorylation
independent

Significance

I propose calpain 2 regulates Akt activity through
cleavage:
 Identified
a novel mechanism for the regulation of
invasion.
Pre-surgery
Glioblastoma
Post-surgery
12 months
17 months
Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.
Acknowledgements
Howard Hughes Medical Institute
 Dr. Kevin Ahern
 Dr. Jeffrey Greenwood
 Greenwood Laboratory Members:

 Sangeet

Lal, M.S.
OSU Biochemistry and Biophysics Department