Regulation of Akt Activity by Calpain 2 in Glioblastoma

Multiform InvasionStudent: Nicholas PihlMentor: Dr. Jeffrey GreenwoodBiochemistry and Biophysics

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Invasion of Glioblastoma Tumor

Progression from Diagnosis to Death in Glioblastoma Patient

Calpain 2 and InvasionCalpain 2

Knockdown Cells Less Invasive

Calpain 2 Cleaves Akt Directly

Determining Effect of Cleavage of Akt

Proposed Mechanism

Ca2+

Calpain 2

PIP3

PIP2PI3K

PTEN

Working Hypothesis

Akt

Cleavage of Akt by Calpain 2Isolated Recombinant and

Native Akt

Akt Treated with Recombinant Calpain 2 and PIP3

Electrophoresis and Western Blotting

Isolating Akt

Immunoprecipitation

Beads Coated with Antibodies

Akt Binds to Beads

Akt is Precipitated and Isolated

Antibody

AktAkt

Bead

Akt Treated with Calpain 2, PIP3

Tubes with Purified Akt

PIP3Calpain 2

Akt

AktAkt Akt

Akt

AktAkt

Akt

Akt

Akt

AktAkt

Bead

Calpain 2 Directly Cleaves rAkt

26 kDa

72 kDa

43 kDa

95 kDa

55 kDa

26 kDa

34 kDa

72 kDa

43 kDa

130 kDa

95 kDa

55 kDa

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

PIP3 Promotes Cleavage of Akt by Calpain 2

0.00

20.00

40.00

60.00

80.00

100.00

120.00

Average Cleavage of Akt by Calpain 2

Per

cen

t

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

26 kDa

17 kDa

34 kDa

72 kDa

55 kDa43 kDa

130 kDa

95 kDa

Calpain 2 Directly Cleaves Native Akt from U87 cells

26 kDa

17 kDa

34 kDa

72 kDa

55 kDa

43 kDa

95 kDa

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

0

20

40

60

80

100

120

Per

cen

t R

emai

nin

g F

ull

Len

gth

A

ktPIP3 Promotes Cleavage of Native Akt by Calpain 2

Contro

l

Calp

ain

2

Capain

2 a

nd

PIP

3

Activation of AktAkt Activated by

Phosphorylation at S473 and Thr308

Hypothesized that Akt Activated by Cleavage

Further Experiments Needed to Test Hypothesis

P P

C-termN-term

Thr308 S473

Catalytic DomainPH Domain

Akt Phosphorylates GSK-3Treating GSK3 with

Full Length Akt

Quantify Amount of GSK-3 Phosphorylated

Akt

GSK-3

PO4

2-

pGSK-3pGSK-3

pGSK-3

More GSK-3 Phosphorylated by Wild-Type Akt

26 kDa

34 kDa

72 kDa

43 kDa

95 kDa

55 kDa

Wild

-Type

Knockd

ow

n

Wild

-Type

lysa

te

Anti-GSK 3

Greater Activity in Wild-Type Calpain 2 Knockdown Cells

0

20

40

60

80

100

120

Rela

tive P

erc

en

tag

es

of

Acti

vit

y

Wild-Type Knockdown

Less Akt Present in Knockdown Samples

26 kDa

34 kDa

72 kDa

43 kDa

55 kDa

Wild

-Typ

e

Knockd

ow

n

Wild

-Typ

e

lysa

tepS473

Wild-Typ

Knockdown

0

20

40

60

80

100

120

Re

lati

ve

Pe

rce

nta

ge

s A

kt

Pre

se

nt

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

Wild-Type Knockdown

ConclusionsCleavage 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