Regulation of Akt Activity by Calpain 2 in Glioblastoma Multiform Invasion

Regulation of Akt Activity by Calpain 2 in Glioblastoma

Multiform InvasionStudent: Nicholas PihlMentor: Dr. Jeffrey GreenwoodBiochemistry 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 InvasionCalpain 2

Knockdown Cells Less Invasive

Calpain 2 Cleaves Akt Directly

Determining Effect of Cleavage of Akt

Proposed Mechanism

Ca2+

Calpain 2

PIP3

PIP2PI3KPTEN

Working Hypothesis

Akt

Cleavage of Akt by Calpain 2Isolated Recombinant and

Native AktAkt Treated with Recombinant

Calpain 2 and PIP3Electrophoresis and Western

Blotting

Isolating AktImmunoprecipitatio

nBeads Coated with

AntibodiesAkt Binds to BeadsAkt is Precipitated

and Isolated

Antibody

AktAkt

Bead

Akt Treated with Calpain 2, PIP3

Tubes with Purified Akt

PIP3Calpain 2Akt

AktAkt Akt

Akt

AktAkt

AktAktAkt

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 kDa95 kDa

55 kDa

Control

Calpain 2

Capain 2 and PIP3

Control

Calpain 2

Capain 2 and PIP3

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

Perc

ent

Control

Calpain 2

Capain 2 and PIP3

26 kDa

17 kDa

34 kDa

72 kDa55 kDa43 kDa

130 kDa95 kDa

Calpain 2 Directly Cleaves Native Akt from U87 cells

26 kDa

17 kDa

34 kDa

72 kDa

55 kDa

43 kDa

95 kDa

Control

Calpain 2

Capain 2 and PIP3

Control

Calpain 2

Capain 2 and PIP3

0

20

40

60

80

100

120

Perc

ent R

emai

ning

Ful

l Len

gth

Akt

PIP3 Promotes Cleavage of Native Akt by Calpain 2

Control

Calpain 2

Capain 2 and PIP3

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 AktQuantify Amount of

GSK-3 Phosphorylated

AktGSK-3

PO42-

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

Knockdown

Wild-Type

lysate

Anti-GSK 3

Greater Activity in Wild-Type Calpain 2 Knockdown Cells

0

20

40

60

80

100

120

Rela

tive

Per

cent

ages

of

Act

ivit

y

Wild-Type Knockdown

Less Akt Present in Knockdown Samples

26 kDa

34 kDa

72 kDa

43 kDa55 kDa

Wild-Type

Knockdown

Wild-Type

lysatepS473

Wild-Typ

Knockdown

0

20

40

60

80

100

120

Rel

ativ

ePer

cent

ages

Akt

Pr

esen

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

Wild-Type Knockdown

ConclusionsCleavage of Akt by Calpain 2

Aided by PIP3Akt from Knockdown Cells More

Active than Wild-TypeFurther Research Needed to

Determine Activity of Breakdown Products

Acknowledgements Howard Hughes Medical InstituteCripps ScholarshipDr. Jeffrey Greenwood, mentorDr. Kevin Ahern, program facilitatorOregon State Biochemistry Department