1. Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion
Student: Nicholas Pihl
Mentor: Dr. Jeffrey Greenwood
Biochemistry and Biophysics

2. Invasion of Glioblastoma Tumor
Progression from Diagnosis to Death in Glioblastoma Patient

3. Calpain 2 and Invasion Calpain 2 Knockdown Cells Less Invasive
Calpain 2 Cleaves Akt Directly
Determining Effect of Cleavage of Akt
Proposed Mechanism
Microenvironment of brain, ECM. 1 micron sized pore, cells are 10 microns wide. Cleaving ECM and distorting shape to navigate these pores.

4. Working Hypothesis Calpain 2 Akt Ca2+ PIP2 PI3K PTEN PIP3
In vitro proteolysis to determine whether Akt is directly cleaved by Calpain 2.
Akt

5. Cleavage of Akt by Calpain 2
Isolated Recombinant and Native Akt
Akt Treated with Recombinant Calpain 2 and PIP3
Electrophoresis and Western Blotting

6. Isolating Akt Akt Akt Bead Immunoprecipitation
Beads Coated with Antibodies
Akt Binds to Beads
Akt is Precipitated and Isolated
Akt
Antibody
Bead

7. Akt Treated with Calpain 2, PIP3
Bead
Tubes with Purified Akt

8. Calpain 2 Directly Cleaves rAkt
130 kDa
95 kDa
95 kDa
72 kDa
72 kDa
55 kDa
55 kDa
43 kDa
43 kDa
34 kDa
26 kDa
26 kDa
Control
Calpain 2
Capain 2 and PIP3
Control
Calpain 2
Capain 2 and PIP3

9. PIP3 Promotes Cleavage of Akt by Calpain 2
Control
Calpain 2
Capain 2 and PIP3

10. Calpain 2 Directly Cleaves Native Akt from U87 cells
95 kDa
130 kDa
72 kDa
95 kDa
55 kDa
72 kDa
55 kDa
43 kDa
43 kDa
34 kDa
34 kDa
26 kDa
26 kDa
17 kDa
17 kDa
Control
Calpain 2
Capain 2 and PIP3
Control
Calpain 2
Capain 2 and PIP3

11. PIP3 Promotes Cleavage of Native Akt by Calpain 2
Control
Calpain 2
Capain 2 and PIP3

12. Activation of Akt Akt Activated by Phosphorylation at S473 and Thr308
Hypothesized that Akt Activated by Cleavage
Further Experiments Needed to Test Hypothesis
Thr308
S473 P P
N-term
C-term
PH Domain
Catalytic Domain

13. Akt Phosphorylates GSK-3
Treating GSK3 with Full Length Akt
Quantify Amount of GSK-3 Phosphorylated
PO4 2-
pGSK-3
Akt
GSK-3
pGSK-3
pGSK-3

14. More GSK-3 Phosphorylated by Wild-Type Akt
Anti-GSK 3
95 kDa
72 kDa
55 kDa
43 kDa
34 kDa
26 kDa
Wild-Type
Wild-Type lysate
Knockdown

15. Greater Activity in Wild-Type Calpain 2 Knockdown Cells

16. Less Akt Present in Knockdown Samples
pS473
72 kDa
55 kDa
43 kDa
34 kDa
26 kDa
Wild-Type lysate
Wild-Type
Knockdown

17. Amount of Full Length Akt in Wild-Type and Knockdown Samples

18. Conclusions Cleavage 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

19. Acknowledgements Howard Hughes Medical Institute Cripps Scholarship
Dr. Jeffrey Greenwood, mentor
Dr. Kevin Ahern, program facilitator
Oregon State Biochemistry Department