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Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion

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.

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Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion

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  1. 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

  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

  4. PIP2 PI3K PTEN PIP3 Working Hypothesis Ca2+ 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 Antibody Bead Immunoprecipitation Beads Coated with Antibodies Akt Binds to Beads Akt is Precipitated and Isolated

  7. Akt Treated with Calpain 2, PIP3 PIP3 Calpain 2 Akt Akt Akt Akt Akt Akt Bead Akt Akt Akt Akt Akt Akt 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 Calpain 2 Control Control Calpain 2 Capain 2 and PIP3 Capain 2 and PIP3

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

  11. PIP3 Promotes Cleavage of Native Akt by Calpain 2 Calpain 2 Control 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 P P S473 Thr308 N-term C-term PH Domain Catalytic Domain

  13. AktPhosphorylates GSK-3 2- • Treating GSK3 with Full Length Akt • Quantify Amount of GSK-3 Phosphorylated PO4 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-TypeCalpain2 Knockdown Cells Knockdown Wild-Type

  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 Wild-Typ Knockdown Wild-Type Knockdown

  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

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