Abstract 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. Materials and Methods 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-D- thiogalactopyranoside) 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. 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 SDS-PAGE 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. Fig. 2 –SDS-PAGE Gel performed with fractions of purified caspase-3 protein after a Ni column Results Marker Conclusion Reverse Primer Forward Primer Site Directed Mutagenesis Select and Purify Clones / Sequence Induction PCR Sonication Protein Purification SDS-PAGE with Coomassie Stain Kinase Assay Caspase-3 Wt PQE31 vector pENTR™/ D-TOPO 2580 bp Restriction Digestion SacI Sequencing Restriction Digestion (Sac1 and Xho1)‏ XhoI pDS- Red pCMV- Flag 4A 4.3 kb Caspase-3 Mutant Fig.2 - Restriction Digestion performed on Caspase-3 mutants with SacI and XhoI enzymes. Regulation of Caspase-3 by PKCδ Sean Crisafi 1, Yadira Malavez 4, and Andrea Doseff 2,3 Bowling Green State University 1, Department of Internal Medicine 2, Department of Molecular Genetics 3, Molecular Cellular and Developmental Biology Program 4, Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, Ohio Caspase-3 and Diagrams Pro- Domain P-17P 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. Future Directions Transfect the mutants into MCF-7 human breast cancer cells to asses their effect on apoptosis. Acknowledgments This work was funded by The Ohio State University’s REU-NSF program, RO1HL NIH, and NSF – MCB (to AID). T0T0 T 30 Sup Pellet Flow Sonic Wash 30 mM 50 mM 100 mM Fig. 3 – Kinase Assay with two phospho-mutants; S12G and S12D Apoptotic Extrinsic signal Receptor Caspase- 9 Caspase- 3 PKCδ P Caspase- 8 C3 Mutant (850bp)‏ Life or Death? XhoI restiction site SacI restriction site MCF-7 cells Instrinsic Apoptotic Signal