Transcript Fig. 2

Regulation of Caspase-3 by PKCδ
Sean
1
Crisafi ,
Yadira
4
Malavez ,
and Andrea
2,3
Doseff
Bowling Green State University1, Department of Internal Medicine2, Department of Molecular Genetics3,
Molecular Cellular and Developmental Biology Program4, Dorothy M. Davis Heart & Lung Research
Institute, The Ohio State University, Columbus, Ohio
Abstract
Receptor
Apoptosis, or programmed cell death, is an essential process
which takes place in a cell. The apoptotic process is activated
when the cell is under stress, infected, or when the genome is
beyond repair. This process is a way for the cell to dispose of itself
in an organized fashion when under these conditions. When the
process is impaired, diseases such as cancer, atherosclerosis and
Parkinson's can occur.
Caspase, a cysteine protease, has a primary role in apoptosis.
One member of the caspase family, caspase-3, is vital in the
apoptotic process. Activation of caspase-3 has been previously
reported to be dependent upon phosphorylation by a kinase,
PKCδ (Protein Kinase C). In order to determine how caspase-3 is
phosphorylated, specific mutants were created to mimic
phosphorylated amino acids and study the effect on caspase-3
activity or activation. Glycine and Aspartic acid mutants were
created in single or multiple amino acid sites. Mutant clones were
introduced into the pENTR-D/TOPO vector, mammalian
expression vectors, and screened by using restriction enzymes,
and then eventually transfected into MCF-7 breast cancer cells to
explore the effect of apoptosis in vivo. Bacterial expression
systems were used to obtain human mutant caspase-3, purified
proteins were used on kinase assays to explore the effect of
phosphorylation in vitro. By utilizing both in vitro and in vivo
approaches and we have learned the role of phosphorylation on
caspase function. These studies increase our understanding of
this unique protease and apoptosis.
Instrinsic
Apoptotic
Signal
Apoptotic
Extrinsic
signal
In vitro kinase assay:
Phosphorylation of human recombinant caspase-3 (rCasp-3) wt
and mutants was achieved by utilizing purified human PKCδ
(Panvera, Invitrogen) as the kinase source. Two hundred and fifty
ng of rCasp-3 was added to 20 µl of kinase reaction mixture
containing 25 mM HEPES, pH 7.4, 10 mM MnCl2, 1 mM MgCl2,
500 nM ATP and 4 Ci of [-32P] ATP in the presence of 10 ng of
human PKC. A mixture of phosphatidylserine (200 g/ml final
concentration) and diacylglycerol (20 g/ml final concentration) was
also added to the reaction as cofactors of PKC.
The
phosphorylation reaction was carried out at 37 °C for 1 h and
was followed by SDS-PAGE. Samples were resolved by SDSPAGE stained with Coomassie.
In vivo (cloning):
PCR was used to amplify the caspase-3 mutant sequence and for
introducing two restriction sites (XhoI and SacI). The mutant
caspase-3 was then cloned into pENTR-D/TOPO vector
(Invitrogen), transformed into competent bacterial cells. DNA was
isolated by QIAPrep Spin Miniprep kit (column DNA). The
caspase-3 mutants, after being cut again with SacI and XhoI,
were cloned into pDS-Red and pCMV-Flag mammalian
expression vectors.
Caspase8
P
500
300
100
Caspase3
Fig.2 - Restriction Digestion performed on Caspase-3
mutants with SacI and XhoI enzymes.
Life or Death?
Forward Primer
Reverse Primer
Caspase-3 Wt
PQE31
vector
Site Directed Mutagenesis
Fig. 2 –SDS-PAGE Gel performed with fractions of purified
caspase-3 protein after a Ni column
Select and Purify Clones / Sequence
PCR
Fig. 3 – Kinase Assay with two phospho-mutants; S12G and S12D
Conclusion
Induction
C3 Mutant (850bp)
SacI restriction site
XhoI restiction site
pENTR™/
D-TOPO
2580 bp
Sonication



Caspase-3 phospho-mimicking mutants S12G and S12D were
generated.
Caspase-3 phospho-mutant proteins were purified in a
bacterial expression system.
Caspase-3 phospho-mutant proteins were utilized in
protein kinase.
Restriction Digestion
SacI
XhoI
Future Directions
Sequencing
Protein Purification
SDS-PAGE
with Coomassie Stain
Restriction Digestion
(Sac1 and Xho1)
Transfect the mutants into MCF-7 human breast cancer cells
to asses their effect on apoptosis.
•
Caspase-3
Mutant
pCMVFlag 4A
4.3 kb
pDSRed
Caspase-3 and Diagrams
Acknowledgments
12 32 36 58 59 67 77
Kinase Assay
P-17
1650
850
Caspase9
Cloning of the Caspase-3 Phospho-Mutants:
Full-length, wild type caspase-3 in a PQE31 vector was used as a
template for the creation of the caspase-3 phospho-mutants using
a Stratagene site-directed mutagenesis kit. The mutants were
used for transforming into bacterial and mammalian expression
vectors.
Protein Purification:
Caspase-3 with a N-terminal His tag was transformed into M15
bacterial cells. A single colony was picked to grow in 3 mL of
Terrific Broth (TB), containing ampicillin and kanamycin as
selective markers, for 8 hours. The culture was then transferred to
20 mL of TB containing 20% glucose and was grown overnight at
37ºC. The culture was then transferred to 1 L of TB and was
grown until it reached an optical density of 0.5 at 37 ºC. The cells
were
induced
with
1mM
IPTG
(isopropyl-β-Dthiogalactopyranoside) for 30 minutes at 20ºC. The culture was
then harvested by centrifugation (6,000 rmp) and the pellet was
resuspended on ice with sonication buffer containing 100 mM
sodium phosphate, 600 mM sodium chloride, 1% Tween, 5 mM
BME, 1mM PMSF, 1 mM Clap. The cells were then sonicated,
breaking the cell wall, to collect the caspase-3 mutant protein. The
lysates were then centrifuged at 12,000 rpm’s. The supernatant
was treated with RNAse and DNAse and incubated with nickel
beads (Ni- NTA-superflow, Qiagen) at 4ºC for 90 min. The
supernatant containing beads were then loaded into a nickel
purifying column, and rinsed with 10 mL of sonication buffer
followed by a rinse with 10 mL of washing buffer. The protein was
later eluted with an Imidazol gradient (30 mM, 50 mM, and
100mM - containing washing buffer 100 mM sodium phosphate,
600 mM sodium chloride, 1% Tween, 10% glycerol, 5 mM BME, 1
mM PMSF, and 1 mM Clap). Finally, the column fractions were
analyzed via SDS-PAGE on 12% acrylamide gel and the proteins
were visualized by Coomassie staining.
3000
PKCδ
Materials and Methods
ProDomain
Results
P-12
MCF-7 cells
This work was funded by The Ohio State University’s
REU-NSF program, RO1HL075040 NIH, and
NSF – MCB0542244 (to AID).