CALPAIN 2 REGULATION OF AKT IN GLIOBLASTOMA CELL INVASION

Presented by Maria NguyenGuidance of Dr. Jeffrey A. Greenwood

Glioblastoma is highly invasive

Less than 5% chance of survival over 5 year span

Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.

Glioblastoma

Pre-surgery Post-surgery 12 months 17 months

Unique mutation in glioma cells

PIP2

PIP3

PTEN

PI3-K

Akt P

INVASION

PTENX

Akt

pAkt localized to membrane projections F-Actin Alexa 555

Unique autocrine glutamate system

INVASION

Calpain 2

Ca2+Ca2+

Ca2+Ca2+

Ca2+Ca2+

Ca2+Ca2+

Ca2+Ca2+

Glutamate

Ca2+Ca2+

Calpain 2 is required for invasion

Jang et al. 2010. Neurochemical Res.

Decreased by

90%

Calpain 2 maintains MMP2 levels

Yamaguchi et al. 2005. Curr. Op. Cell Biol. 17: 559-564.

Actin

Matrix metalloproteinase 2(MMP2)

What are the mechanisms of regulation?

Calpain 2

Akt

MMP2

Invasion

MMP2 and Akt: Akt involved in the expression

of MMP2

? Either…

1. Directly2. Indirectly

Testing pAkt levels

72kDa

55kDa

43kDa

pAkt (Ser-473)

Actin

0

0.4

0.8

1.2

1.6

ControlKD

UG87MG cell lines

pAkt

/A

ctin

Control KD

Time dependent dephosphorylation

70kDa

55kDa

70kDa

55kDa

Control

KD

SD 0’ 5’ 15’ 30’ 60’

0 20 40 600

20

40

60

80

100

ControlKD

Time

% o

f con

trol

LY294002

PIP2

PIP3

PI3-K

Akt P

Akt

XX

Calpain 2 regulates proteolysis of Akt

70kDa

55kDa

25kDa

Control KD

70kDa100kDa

Calpain 2

pAkt (Ser-473)

BDPs

U87MG cell lines0

10

20

30

40

50

ControlKD

% o

f tot

al A

kt

Working Hypothesis

Calpain 2 directly cleaves Akt in the regulation of glioblastoma cell invasion.

Calpain 2

Ca2+Ca2+

Calpain 2

Ca2+Ca2+

P P

P

P

PPP

Akt unfolds and is susceptible to cleavage

N-term C-term

PIP3

Proposed site of cleavage

Site of cleavage for a 22 kDa fragment is estimated to lie in the middle of the catalytic domain

About 296

PHdomain

Catalyticdomain

Regulatory domain

Thr 308 Ser 473

N-term

P P

C-term

N-term

Catalyticdomain

PHdomain

Calpain 2 cleavage of Akt could mean:1. Inactivation2. Phosphorylation independent activation

PIP3

Significance

I propose calpain 2 regulates Akt activity through cleavage: Identified a novel mechanism for the regulation of

invasion.

Nakada et al. 2007. Cell. Mol. Life Sci. 64: 458-478.Glioblastoma

Pre-surgery Post-surgery 12 months 17 months

Acknowledgements

Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Jeffrey Greenwood Greenwood Laboratory Members:

Sangeet Lal, M.S. OSU Biochemistry and Biophysics Department