MATERIALS AND METHODS

2.2.1 Bioinformatics and database searching

We designed a Perl based peptide match program to search the human proteome databases (Swiss-Prot and National Center for Biotechnology Information, 07/10/12) for overlapping CK2 and caspase consensus sequences. Overlapping motifs were designed such that the putative phospho-acceptor (S/T-X-X-D) is positioned at P2 or P1" of the predicted scissile bond for caspase-3/-7 (D-E-X-D) and caspase-8/-9 (I/V/L-E-X-D). After a proteome-wide search for matching peptides was performed, the peptide match

Prot protein ID, as well as the sequence of the matching peptide (±10 amino acid residues on either side of the recognized peptide pattern). Subsequently, database mining was performed by manually searching a caspase substrate database (CASBAH) (20), and phosphorylation databases (Phosida and UniProt KB) (21) for the predicted CK2/caspase modifications.

2.2.2 Generation and purification of caspase-3 and related mutants

Catalytically inactive His-tagged caspase-3 (ATCC) was engineered using a QuickChange II Site-Directed Mutagenesis Kit (Stratagene). Human caspase-3 pET23b was PCR amplified to make the (C163A) mutation to abolish catalytic activity thereby allowing for purification of full-length caspase-3 using the primer 5’- TTCATTATTCAGGCCGCCCGTGGTACAGAA-3’. In all cases, recombinant caspase-3 used in this study contains C163A mutation and, therefore, is the full-length, zymogen form. Phosphorylation mutants (T174A), (S176A) and (T174A S176A) of caspase-3 were generated using the following primers 5’-GACTGTGGCATTGAGGCAGACAGTGGTGGTGAT-

3’_, 5’-_{GGCATTGAGACAGACGCTGGTGGTGATGAC-}3’_, 5’_-

GACTGTGGCATTGAGGCAGACGCTGGTGTTGATGATGAC-3’, respectively. caspase-3 (C163A) and the phosphorylation mutants were purified with a HiTrap SP HP column (Amersham Biosciences) using the AKTA Purifier FPLC (Amersham Biosciences). Purified, recombinant caspase was then dialyzed into 50% glycerol, 50 mM Tris (tris(hydroxymethyl)aminomethane) (pH 7.5), and 150 mM NaCl and stored at -20°C.

In vitro kinase assays were performed using recombinant, 6!His-tagged, full- length, inactive (C163A) caspase-3. For phosphorylation, 0.5 – 2 µg of recombinant caspase was incubated with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1 mM ATP, 0.2 µCi ["-32P]-ATP and 35 ng of recombinant GST-CK2# for 20 min at 30°C. Assays investigating stoichiometry of phosphorylation or being used for downstream 2D- peptide mapping were allowed to run for 2 h. Reactions were stopped by the addition of SDS sample buffer and proteins resolved by 10% SDS-PAGE gel electrophoresis. Gels were then dried and radiolabeled proteins visualized using a phosphorimager. The relative amount of 32P incorporation was determined using ImageQuant TL software.

For kinetic analysis of caspase-3, calmodulin and #-casein, 0.5 – 8 µM purified protein substrate was reacted with CK2 and terminated by adding 2! SDS sample buffer. Reactions were performed for 5 and 10 minutes to verify linearity. The holoenzyme was reconstituted by incubating equimolar amounts of His-CK2$ and GST-CK2# for 5 minutes prior to the assay. Samples were processed by SDS-PAGE, stained with coommassie blue and then dried. Phosphorylation was detected using a phosphorimager and absolute 32P incorporation was determined using ImageQuant TL software. Kinetic constants were determined using GraphPad Prism software version 4.0.

2.2.4 2D phospho-peptide mapping and phospho-amino acid analysis

2D phospho-peptide mapping was performed as previously described (22). Briefly, reactions were resolved by SDS-PAGE, the gel dried and bands corresponding to caspase 3 were excised. Proteins were digested by trypsin overnight and then separated

chromatography in Scheidtmann buffer (vertical dimension). Plates were analyzed by a phosphorimager. Next, the indicated spots were scraped off the plate and processed for phospho-amino acid analysis. Briefly, recovered peptides were partially hydrolyzed in constant boiling HCl as previously described (23). Next, two-dimensional separation of partially hydrolyzed products was carried out as previously described (24). Briefly, phospho-amino acids were spotted on thin layer chromatography plates and separated in pH 1.9 buffer (2.5% formic acid/7.8% glacial acetic acid) in the first dimension (horizontal in figures) and pH 3.5 buffer (0.5% pyridine/5.0% glacial acetic acid) in the second. 32P-amino acids were visualized using a phosphorimager and the presence or absence of phospho-amino acids was determined empirically.

2.2.5 Cleavage of caspase-3 by caspase-8 and -9

To test the effect of caspase-3 phosphorylation on caspase-mediated cleavage, 2 µg of purified caspase-3 were incubated with GST-CK2! in a kinase assay for 2 h as described above. Following the kinase assay, 6 U/µl of caspase-8 (BIOMOL) or caspase- 9 (BIOMOL) was added to the kinase reaction in caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT) for caspase-8 or (100 mM MES, pH 6.5, 10% PEG (Polyethylene Glycol, average MW 8000), 0.1% CHAPS, 10 mM DTT) for caspase-9 reactions. Cleavage reactions were incubated overnight at 30 0C with gentle agitation to obtain optimal cleavage and then stopped by addition of SDS sample buffer. Radiolabeled proteins were resolved on 12% SDS-PAGE electrophoresis, and gels were dried and visualized on a phosphorimager. Determination of the amount of 32P incorporated was assessed using ImageQuant TL

software. Non-radiolabeled cleavage reactions were performed in parallel to verify caspase-8 and -9 activity. Cold kinase assays (non-radiolabelled ATP) were performed as above except were resolved on 12% SDS-PAGE, transferred to PVDF and probed with anti-caspase-3 antibodies.

2.2.6 Western blotting

Samples were separated by SDS-PAGE and transferred to PVDF using standard procedures. Antibodies against caspase-3 (Cell Signaling Technology) and Phospho- threonine (Cell Signaling Technology) were used. Phospho-specific caspase-3 antibodies were generated against GIEpTDpSGVDDMAC by YenZym Antibodies, LLC, San Francisco, Ca using proprietary methods. Secondary antibodies used were GAR-680 (LI- COR Biosciences). Densitometric analysis was performed using Odyssey software (version 3.0) from LI-COR Biosciences.