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Calpain 2 regulation of Akt in glioblastoma cell invasion

Calpain 2 regulation of Akt in glioblastoma cell invasion. Presented by Maria Nguyen Guidance of Dr. Jeffrey A. Greenwood. Glioblastoma is highly invasive. Less than 5% chance of survival over 5 year span. Pre-surgery. Post-surgery. 12 months. 17 months. Glioblastoma.

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Calpain 2 regulation of Akt in glioblastoma cell invasion

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  1. Calpain 2 regulation of Aktin glioblastoma cell invasion Presented by Maria Nguyen Guidance of Dr. Jeffrey A. Greenwood

  2. Glioblastoma is highly invasive • Less than 5% chance of survival over 5 year span Pre-surgery Post-surgery 12 months 17 months Glioblastoma Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.

  3. Unique mutation in glioma cells PIP2 X PTEN PTEN PI3-K P PIP3 Akt Akt INVASION

  4. pAkt localized to membrane projections F-Actin Alexa 555

  5. Glutamate Unique autocrine glutamate system Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Calpain 2 INVASION

  6. Calpain 2 is required for invasion Decreased by 90% Jang et al. 2010. Neurochemical Res.

  7. Calpain 2 maintains MMP2 levels Actin Matrix metalloproteinase 2 (MMP2) Yamaguchi et al. 2005. Curr. Op. Cell Biol. 17: 559-564.

  8. What are the mechanisms of regulation? • MMP2 and Akt: • Akt involved in the expression of MMP2 Calpain 2 • Either… • Directly • Indirectly ? Akt MMP2 Invasion

  9. Testing pAkt levels 72kDa pAkt (Ser-473) 55kDa Actin 43kDa Control KD

  10. Time dependent dephosphorylation LY294002 SD 0’ 5’ 15’ 30’ 60’ 70kDa Control 55kDa 70kDa KD 55kDa P PIP2 Akt X PI3-K PIP3 X Akt

  11. Calpain 2 regulates proteolysis of Akt Control KD 100kDa Calpain 2 70kDa 70kDa pAkt (Ser-473) 55kDa 25kDa BDPs

  12. Working Hypothesis Calpain 2 directly cleaves Akt in the regulation of glioblastoma cell invasion. Ca2+ Ca2+ Ca2+ Ca2+ Calpain 2 Calpain 2

  13. Akt unfolds and is susceptible to cleavage P P P P P P P P PIP3 N-term C-term

  14. Proposed site of cleavage • Calpain 2 cleavage of Akt could mean: • Inactivation • Phosphorylation independent activation PH domain Catalytic domain PH domain Catalytic domain Regulatory domain Ca2+ Thr 308 Ser 473 Ca2+ P P Calpain 2 N-term N-term C-term PIP3 About 296 Site of cleavage for a 22 kDa fragment is estimated to lie in the middle of the catalytic domain

  15. Significance • I propose calpain 2 regulates Akt activity through cleavage: • Identified a novel mechanism for the regulation of invasion. Pre-surgery Post-surgery 12 months 17 months Glioblastoma Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.

  16. Acknowledgements • Howard Hughes Medical Institute • Dr. Kevin Ahern • Dr. Jeffrey Greenwood • Greenwood Laboratory Members: • Sangeet Lal, M.S. • OSU Biochemistry and Biophysics Department

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