Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner

Abstract

Caspase activation in target cells is a major function of granzyme B (grB) during cytotoxic lymphocyte granule-induced apoptosis. grB-mediated cell death can occur in the absence of active caspases, and the molecular targets responsible for this additional pathway remain poorly defined. Apoptotic plasma membrane blebbing is caspase independent during granule exocytosis–mediated cell death, whereas in other instances, this event is a consequence of the cleavage by caspases of the Rho effector, Rho-associated coiled coil–containing protein kinase (ROCK) I. We show here that grB directly cleaves ROCK II, a ROCK family member encoded by a separate gene and closely related to ROCK I, and this causes constitutive kinase activity and bleb formation. For the first time, two proteins of the same family are found to be specifically cleaved by either a caspase or grB, thus defining two independent pathways with similar phenotypic consequences in the cells. During granule-induced cell death, ROCK II cleavage by grB would overcome, for this apoptotic feature, the consequences of deficient caspase activation that may occur in virus-infected or malignant target cells.

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

“…30,31 Alternatively, caspase-7 or other caspases may synergize with gzmB in targeting and perturbing the cytoskeleton by cleaving a-tubulin, the tubulin folding cofactor and/or other relevant proteins, such as filamin and ROCKII. 14,15,32 However, the data do not exclude the Figure 7 Model for gzmB-mediated pro-apoptotic pathways and cell death elicited by ex vivo CTL. gzmB exerts pleiotropic pro-apoptotic potential in gzmB þ CTL-induced cell death.…”

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

“…However, this athetotic morphology was accompanied by some residual membrane blebbing, most likely due to GzmB directly inducing blebbing without caspase activation. 19 Although morphologically and kinetically distinct from necrosis, we also tested whether athetosis may converge on the programmed necrosis/necroptosis pathway, which requires RIP1. 20 However, treatment with the RIP1 inhibitor necrostatin-1 did not inhibit GzmB À / À NK cell-mediated death, either in the presence or absence of QVD (Supplementary Figure S3D).…”

Rho kinase in the regulation of cell death and survival

“…The nature of the proteases involved and the cleavage site for caspase 3-independent cleavage of ROCK1 remain to be identified [17,91]. On the other hand, during cytotoxic lymphocyte granule-induced cell death, human ROCK2 can be cleaved by the proapoptotic protease granzyme B at IGLD1131 site, and this site is not present in ROCK1 [118]. Human ROCK2, but not ROCK1, can also be activated by caspase 2-dependent cleavage in endothelial cells in response to thrombin, but the cleavage site remains to be identified [117].…”

“…An apoptotic role for other ROCK activation pathway such as Rho/ROCK [59,89], granzyme B/ROCK2 [118], caspase 2/ROCK2 [117] may be highly cell type-dependent and/or apoptotic stimulus-dependent. Importantly, ROCK1 and ROCK2 can be distinctly activated or inhibited by a number of positive or negative regulators, and in turn can have distinct cellular or physiological functions.…”

“…Finally, ROCK2, but not ROCK1, can be activated by granzyme B-mediated cleavage (at Asp 1131) in a context of cytotoxic lymphocyte granule-induced apoptosis. This cleaved (activated) ROCK2 is able to induce membrane blebbing independent of caspase 3 activation [118].…”