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 Progression from Diagnosis to Death in Glioblastoma Patient

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.

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

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

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

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

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

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

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

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

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

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

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

Greater Activity in Wild-Type Calpain 2 Knockdown Cells

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

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

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

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