regulation of akt activity by calpain 2 in glioblastoma multiform invasion student: nicholas pihl...
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Regulation of Akt Activity by Calpain 2 in Glioblastoma
Multiform InvasionStudent: Nicholas PihlMentor: Dr. Jeffrey GreenwoodBiochemistry and Biophysics
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Calpain 2 and InvasionCalpain 2
Knockdown Cells Less Invasive
Calpain 2 Cleaves Akt Directly
Determining Effect of Cleavage of Akt
Proposed Mechanism
Cleavage of Akt by Calpain 2Isolated Recombinant and
Native Akt
Akt Treated with Recombinant Calpain 2 and PIP3
Electrophoresis and Western Blotting
Isolating Akt
Immunoprecipitation
Beads Coated with Antibodies
Akt Binds to Beads
Akt is Precipitated and Isolated
Antibody
AktAkt
Bead
Akt Treated with Calpain 2, PIP3
Tubes with Purified Akt
PIP3Calpain 2
Akt
AktAkt Akt
Akt
AktAkt
Akt
Akt
Akt
AktAkt
Bead
Calpain 2 Directly Cleaves rAkt
26 kDa
72 kDa
43 kDa
95 kDa
55 kDa
26 kDa
34 kDa
72 kDa
43 kDa
130 kDa
95 kDa
55 kDa
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
PIP3 Promotes Cleavage of Akt by Calpain 2
0.00
20.00
40.00
60.00
80.00
100.00
120.00
Average Cleavage of Akt by Calpain 2
Per
cen
t
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
26 kDa
17 kDa
34 kDa
72 kDa
55 kDa43 kDa
130 kDa
95 kDa
Calpain 2 Directly Cleaves Native Akt from U87 cells
26 kDa
17 kDa
34 kDa
72 kDa
55 kDa
43 kDa
95 kDa
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
0
20
40
60
80
100
120
Per
cen
t R
emai
nin
g F
ull
Len
gth
A
ktPIP3 Promotes Cleavage of Native Akt by Calpain 2
Contro
l
Calp
ain
2
Capain
2 a
nd
PIP
3
Activation of AktAkt Activated by
Phosphorylation at S473 and Thr308
Hypothesized that Akt Activated by Cleavage
Further Experiments Needed to Test Hypothesis
P P
C-termN-term
Thr308 S473
Catalytic DomainPH Domain
Akt Phosphorylates GSK-3Treating GSK3 with
Full Length Akt
Quantify Amount of GSK-3 Phosphorylated
Akt
GSK-3
PO4
2-
pGSK-3pGSK-3
pGSK-3
More GSK-3 Phosphorylated by Wild-Type Akt
26 kDa
34 kDa
72 kDa
43 kDa
95 kDa
55 kDa
Wild
-Type
Knockd
ow
n
Wild
-Type
lysa
te
Anti-GSK 3
Greater Activity in Wild-Type Calpain 2 Knockdown Cells
0
20
40
60
80
100
120
Rela
tive P
erc
en
tag
es
of
Acti
vit
y
Wild-Type Knockdown
Less Akt Present in Knockdown Samples
26 kDa
34 kDa
72 kDa
43 kDa
55 kDa
Wild
-Typ
e
Knockd
ow
n
Wild
-Typ
e
lysa
tepS473
Wild-Typ
Knockdown
0
20
40
60
80
100
120
Re
lati
ve
Pe
rce
nta
ge
s A
kt
Pre
se
nt
Amount of Full Length Akt in Wild-Type and Knockdown Samples
Wild-Type Knockdown
ConclusionsCleavage of Akt by Calpain 2
Aided by PIP3
Akt from Knockdown Cells More Active than Wild-Type
Further Research Needed to Determine Activity of Breakdown Products
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