Identification of {alpha}-tubulin as a granzyme B substrate during CTL-mediated apoptosis.
Cytotoxic lymphocytes induce target cell apoptosis via two major pathways: Fas/FasL and granule exocytosis. The latter pathway has largely been defined by the roles of the pore-forming protein perforin and by the serine proteinases granzymes A and B. Upon entry into target cells, the granzymes cleave substrates that ultimately result in cell death. To gain further insight into granzyme B function, we have identified novel substrates. SDS-PAGE analysis of S100 cell lysates identified a 51 kDa protein that was cleaved by granzyme B. Mass spectrometry analysis revealed that this fragment was the microtubule protein, alpha-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of alpha-tubulin had a more basic isoelectric point than the full-length molecule, suggesting that granzyme B removed the acidic C-terminus. Site-directed mutagenesis within this region of alpha-tubulin revealed the granzyme B recognition site, which is conserved in a subset of alpha-tubulin isoforms. Significantly, we showed that alpha-tubulin was cleaved in target cells undergoing apoptosis as induced by cytotoxic T lymphocytes. Therefore, in addition to its role in the activation of mitochondria during apoptosis, these results suggest a role for granzyme B in the dismantling of the cytoskeleton.
Goping
Ing Swie
IS
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Sawchuk
Tracy
T
Underhill
D Alan
DA
Bleackley
R Chris
RC
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D053804:Granzymes; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D020033:Protein Isoforms; D014404:Tubulin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases