Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion
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Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion Student: Nicholas Pihl Mentor: Dr. Jeffrey Greenwood Biochemistry and Biophysics http://www.lifesciences.sourcebioscience.com/media/274486/cell%20cultur
description
Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion. Student: Nicholas Pihl Mentor: Dr. Jeffrey Greenwood Biochemistry and Biophysics. http://www.lifesciences.sourcebioscience.com/media/274486/cell%20culture.jpg. Invasion of Glioblastoma Tumor. - PowerPoint PPT Presentation
Transcript of Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion
Regulation of Akt Activity by Calpain 2 in Glioblastoma
Multiform InvasionStudent: Nicholas PihlMentor: Dr. Jeffrey GreenwoodBiochemistry and Biophysics
http://www.lifesciences.sourcebioscience.com/media/274486/cell%20culture.jpg
Invasion of Glioblastoma Tumor
Progression from Diagnosis to Death in Glioblastoma Patient
Calpain 2 and InvasionCalpain 2
Knockdown Cells Less Invasive
Calpain 2 Cleaves Akt Directly
Determining Effect of Cleavage of Akt
Proposed Mechanism
Ca2+
Calpain 2
PIP3
PIP2PI3KPTEN
Working Hypothesis
Akt
Cleavage of Akt by Calpain 2Isolated Recombinant and
Native AktAkt Treated with Recombinant
Calpain 2 and PIP3Electrophoresis and Western
Blotting
Isolating AktImmunoprecipitatio
nBeads Coated with
AntibodiesAkt Binds to BeadsAkt is Precipitated
and Isolated
Antibody
AktAkt
Bead
Akt Treated with Calpain 2, PIP3
Tubes with Purified Akt
PIP3Calpain 2Akt
AktAkt Akt
Akt
AktAkt
AktAktAkt
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 kDa95 kDa
55 kDa
Control
Calpain 2
Capain 2 and PIP3
Control
Calpain 2
Capain 2 and PIP3
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
Perc
ent
Control
Calpain 2
Capain 2 and PIP3
26 kDa
17 kDa
34 kDa
72 kDa55 kDa43 kDa
130 kDa95 kDa
Calpain 2 Directly Cleaves Native Akt from U87 cells
26 kDa
17 kDa
34 kDa
72 kDa
55 kDa
43 kDa
95 kDa
Control
Calpain 2
Capain 2 and PIP3
Control
Calpain 2
Capain 2 and PIP3
0
20
40
60
80
100
120
Perc
ent R
emai
ning
Ful
l Len
gth
Akt
PIP3 Promotes Cleavage of Native Akt by Calpain 2
Control
Calpain 2
Capain 2 and PIP3
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 AktQuantify Amount of
GSK-3 Phosphorylated
AktGSK-3
PO42-
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
Knockdown
Wild-Type
lysate
Anti-GSK 3
Greater Activity in Wild-Type Calpain 2 Knockdown Cells
0
20
40
60
80
100
120
Rela
tive
Per
cent
ages
of
Act
ivit
y
Wild-Type Knockdown
Less Akt Present in Knockdown Samples
26 kDa
34 kDa
72 kDa
43 kDa55 kDa
Wild-Type
Knockdown
Wild-Type
lysatepS473
Wild-Typ
Knockdown
0
20
40
60
80
100
120
Rel
ativ
ePer
cent
ages
Akt
Pr
esen
tAmount of Full Length Akt in Wild-Type and Knockdown Samples
Wild-Type Knockdown
ConclusionsCleavage of Akt by Calpain 2
Aided by PIP3Akt from Knockdown Cells More
Active than Wild-TypeFurther Research Needed to
Determine Activity of Breakdown Products
Acknowledgements Howard Hughes Medical InstituteCripps ScholarshipDr. Jeffrey Greenwood, mentorDr. Kevin Ahern, program facilitatorOregon State Biochemistry Department
