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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02791

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Identification of -tubulin as a granzyme B substrate during CTL-mediated apoptosis

¹ Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7
² Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7

View larger version (44K): [in a new window]   Fig. 1. Coomassie-Blue-stained gel of HeLa S100 extracts. 30 µg HeLa S100 extract were left untreated (lane 3) or treated with 5 µM and 50 µM purified grB in a 10 µl reaction volume (lanes 4-5, 7-8). 100 µM zVAD-fmk was added to lanes 6-8. Protein products were separated by a 12% SDS-PAGE and visualized by staining with Coomassie Blue R250. Positions of molecular size markers (in kDa) are indicated on the left.

View larger version (37K): [in a new window]   Fig. 2. Amino acid sequence of -tubulin and western blot analysis of -tubulin from S100 cell extracts. (A) The amino acid sequence of human -tubulin (TUBA3). Solid boxes indicated the tryptic peptides that were identified by mass spectrometry analysis. Arrows indicate putative grB cleavage sites. (B) HeLa S100 (lanes 1-6) or Jurkat S100 (lanes 7-12) were treated with the indicated amounts of purified grB (in a 10 µl reaction volume), in the presence or absence of 100 µM zVAD-fmk. Protein products were separated by 12% SDS-PAGE and transferred to nitrocellulose. The migration of -tubulin (upper) and caspase 3 (lower) were assessed by western blot analysis. Full-length -tubulin is indicated by *, and cleaved -tubulin by **. GrB-dependent processing of pro-caspase 3 is indicated by p20, and caspase-dependent processing is indicated by p19.

View larger version (32K): [in a new window]   Fig. 3. Two-dimensional western blot of -tubulin from S100 extracts and western blot analysis of -tubulin produced from transcription/translation reactions. (A) Jurkat S100 (30 µg) was incubated with or without purified grB (50 µM), as indicated. The reactions were then separated on a first dimension IEF IPGphor strip with pH range of 4-6. The second dimension was 12% SDS-PAGE and the presence of -tubulin was determined by western blotting with anti--tubulin. (B) -tubulin containing FLAG-tagged wild-type amino acid sequence (WT) or point mutants D424A, D431A or D438A, as indicated were created by TNT reactions of the corresponding plasmids. Reactions were incubated with (+) or without (–) purified grB (50 µM). Protein products were separated on 12% SDS-PAGE for western blot analysis with anti-FLAG.

View larger version (25K): [in a new window]   Fig. 4. Western blot analysis of -tubulin from apoptotic lysates. (A) HeLa cells (lanes 4-11) or Jurkat cells (lanes 15-22) were incubated with CTLs at increasing effector-to-target ratio (E:T) (0.5:1, 1:1 and 2:1) for 4 hours at 37°C. EGTA was included to inhibit granular-mediated killing (lanes 8-11, 19-22). Protein products were separated by 12% SDS-PAGE and analyzed by western blot with anti -tubulin. Full-length -tubulin is indicated by *, and cleaved -tubulin by **. (B) Jurkat cells were incubated with CTLs at increasing E:T (2:1, 5:1 and 10:1) for 4 hours at 37°C. zVAD-fmk (100 µM final concentration) was included to inhibit caspase activation (lanes 5-8). Protein products were separated by 10% SDS-PAGE and analyzed by western blot with anti -tubulin (upper panel) and anti-caspase 3 (lower panel). GrB-dependent processing of pro-caspase 3 is indicated by p20, and caspase-dependent processing is indicated by p19 and p17.

View larger version (36K): [in a new window]   Fig. 5. Immunoprecipitation of -tubulin from apoptotic lysates. (A) Jurkat cells pre-labelled with [35S]methionine were incubated with unlabelled effector cells at an E:T of 1:1. Specific full-length -tubulin is indicated by * in lane 1, and cleaved product by ** in lane 2. Non-specific products are shown in lanes 3 and 4. (B) Jurkat cell lysates were separated into fractions containing soluble -tubulin (S) and fractions containing polymerized microtubules (P) (see Materials and Methods for details). GrB was added to 5 µM (lanes 2 and 5) and 10 µM (lanes 3 and 6). Protein products were separated by 10% SDS-PAGE and analyzed by western blot with anti -tubulin. (C) Jurkat and CTL (J + CTL) were incubated at an E:T of 1:1 prior to solubilization. The total protein solution (T) was separated into fractions containing soluble -tubulin (S) and fractions containing polymerized microtubules (P) (lanes 10-12). The efficiency of fractionation to the soluble phase was verified by incubation of Jurkat cells with the microtubule depolymerizing agent nocodazole (Noc, lanes 4-6). The efficiency of fractionation to the polymerized phase was verified by incubation of Jurkat cells with the microtubule polymerizing agent paclitaxel (Pac, lanes 7-9). Protein products were separated on a 12% SDS-PAGE and analyzed by western blot with anti -tubulin.

View larger version (32K): [in a new window]   Fig. 6. Sequence alignment of -tubulin isoforms. The C-terminal portion of 11 human -tubulin isoforms is shown (full-length sequences are available in Fig. S1 in supplementary material). Amino acid identity is indicated with black highlighting, with the exception of the grB cleavage site, which is highlighted in grey; mutation of this site (indicated by an arrow) was found to ablate grB cleavage in vitro. The -tubulin isoforms include those previously described in the literature, together with additional proteins annotated in the ENSEMBL Genome Browser (www.ensembl.org; protein family ENSF00000000220) that could be independently verified as expressed sequence tags through the NCBI Blast server (www.ncbi.nlm.nih.gov/BLAST). Alignments were generated using ClustalW and the length of each isoform is indicated in brackets.