CALPAIN 2 REGULATION OF AKT IN GLIOBLASTOMA CELL INVASION Presented by Maria Nguyen Guidance of Dr....
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Transcript of CALPAIN 2 REGULATION OF AKT IN GLIOBLASTOMA CELL INVASION Presented by Maria Nguyen Guidance of Dr....
CALPAIN 2 REGULATION OF AKT IN GLIOBLASTOMA CELL INVASION
Presented by Maria NguyenGuidance of Dr. Jeffrey A. Greenwood
Glioblastoma is highly invasive
Less than 5% chance of survival over 5 year span
Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.
Glioblastoma
Pre-surgery Post-surgery 12 months 17 months
Unique mutation in glioma cells
PIP2
PIP3
PTEN
PI3-K
Akt P
INVASION
PTENX
Akt
pAkt localized to membrane projections F-Actin Alexa 555
Unique autocrine glutamate system
INVASION
Calpain 2
Ca2+Ca2+
Ca2+Ca2+
Ca2+Ca2+
Ca2+Ca2+
Ca2+Ca2+
Glutamate
Ca2+Ca2+
Calpain 2 is required for invasion
Jang et al. 2010. Neurochemical Res.
Decreased by
90%
Calpain 2 maintains MMP2 levels
Yamaguchi et al. 2005. Curr. Op. Cell Biol. 17: 559-564.
Actin
Matrix metalloproteinase 2(MMP2)
What are the mechanisms of regulation?
Calpain 2
Akt
MMP2
Invasion
MMP2 and Akt: Akt involved in the expression
of MMP2
? Either…
1. Directly2. Indirectly
Testing pAkt levels
72kDa
55kDa
43kDa
pAkt (Ser-473)
Actin
0
0.4
0.8
1.2
1.6
ControlKD
UG87MG cell lines
pAkt
/A
ctin
Control KD
Time dependent dephosphorylation
70kDa
55kDa
70kDa
55kDa
Control
KD
SD 0’ 5’ 15’ 30’ 60’
0 20 40 600
20
40
60
80
100
ControlKD
Time
% o
f con
trol
LY294002
PIP2
PIP3
PI3-K
Akt P
Akt
XX
Calpain 2 regulates proteolysis of Akt
70kDa
55kDa
25kDa
Control KD
70kDa100kDa
Calpain 2
pAkt (Ser-473)
BDPs
U87MG cell lines0
10
20
30
40
50
ControlKD
% o
f tot
al A
kt
Working Hypothesis
Calpain 2 directly cleaves Akt in the regulation of glioblastoma cell invasion.
Calpain 2
Ca2+Ca2+
Calpain 2
Ca2+Ca2+
P P
P
P
PPP
Akt unfolds and is susceptible to cleavage
N-term C-term
PIP3
Proposed site of cleavage
Site of cleavage for a 22 kDa fragment is estimated to lie in the middle of the catalytic domain
About 296
PHdomain
Catalyticdomain
Regulatory domain
Thr 308 Ser 473
N-term
P P
C-term
N-term
Catalyticdomain
PHdomain
Calpain 2 cleavage of Akt could mean:1. Inactivation2. Phosphorylation independent activation
PIP3
Significance
I propose calpain 2 regulates Akt activity through cleavage: Identified a novel mechanism for the regulation of
invasion.
Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.Glioblastoma
Pre-surgery Post-surgery 12 months 17 months
Acknowledgements
Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Jeffrey Greenwood Greenwood Laboratory Members:
Sangeet Lal, M.S. OSU Biochemistry and Biophysics Department