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

Abstract

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, α-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of α-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 α-tubulin revealed the granzyme B recognition site, which is conserved in a subset of α-tubulin isoforms. Significantly, we showed that α-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.

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs

“…13 For the second mechanism, gzmB was found to directly cleave a number of structural and regulatory proteins of the cytoskeleton, cytosol and nucleus. 4,5,8,[14][15][16][17] This constitutes a caspase-independent gzmB trigger of CTL-mediated cell death, but the contribution of each substrate cleavage event to cell death remains poorly defined. A notable exception is the BH3-only protein Bid.…”

A ‘polarized’ look at α-tubulin cleavage by granzyme B

“…This polarity allows for a process known as 'dynamic instability' in which the positive end is extended by a net addition of subunits with a concurrent net loss of subunits at the negative end. Adrain et al 13 and Goping et al, 14 demonstrated that granzyme B-cleaved a-tubulin at Asp438, thereby removing a short sequence of acidic amino acids (SVEGEGEEEGEEEY) from the C-terminus. As the polarized nature of a-tubulin contributes to the dynamic nature of microtubules, removal of Cell Death and Differentiation (2006) 13, 1839-1841 these acidic (negative) residues is likely to impact on microtubule stability.…”

“…While we suggest that more rigorous tests should have been carried out to verify that a-tubulin is a bona-fide physiological substrate for granzyme B, these studies leave us with little doubt that human and murine granzyme B have the ability to cleave a-tubulin in cell lysates 8,13,14 and that atubulin is cleaved in a caspase-independent manner in target cells killed by cytotoxic lymphocytes 13,14 strongly suggesting that a-tubulin is cleaved by granzyme B in cells. It is therefore intriguing to speculate why granzyme B cleaves a-tubulin and what consequences this has for the target cell.…”

“…Granzyme B is then delivered to the target cell cytosol by a perforin-dependent mechanism. Adrain et al, 13 showed that a-tubulin was cleaved in a caspase-independent manner in HeLa cells that were killed by the NK cell line YT, while Goping et al 14 showed similar results using CTL stimulated with Epstein Barr virus transformed Rosewell's Park Memorial Institute media (RPMI)-8666 cells. In assays using intact NK (YT) or CTL (RPMI-8666) it is difficult to discount a potential role of other granzymes, delivered to the target cell along with granzyme B, in cleaving a-tubulin.…”

“…Using this approach they have noted 15 potential substrates for granzyme B. A similar approach was employed by Goping et al 14 in which protein fragments from granzyme B-treated lysates were resolved by conventional 1D-PAGE and identified by mass spectroscopy. Both groups identified a-tubulin as a substrate for human granzyme B, supporting a previous report which showed that a-tubulin was cleaved in lysates from YAC-1 (murine lymphoma) cells treated with murine granzyme B.…”