We have demonstrated that ROCK II is cleaved by grB during granule-mediated killing of target cells. ROCK II cleavage by grB removes an inhibitory domain similar to that deleted in ROCK I by caspase-3 cleavage (7
). Consequently, the NH2
-terminal fragment of ROCK II contains the entire kinase domain, is constitutively active, and is sufficient to trigger MLC phosphorylation and plasma membrane blebbing in a Rho-independent manner. Therefore, the consequences of ROCK II cleavage by grB are similar to that of ROCK I cleavage by caspases. This is the first time that two proteins of a same family, with no functional differences, are found to be specifically cleaved by either a caspase or grB, thus defining two specific pathways with similar phenotypic consequences in the cells.
Several substrates common to caspases and grB have been previously identified. Most often, grB and caspase-3 process these common substrates at different sites, usually within an ~15–amino acid span, thus generating distinct cleavage fragments. With few exceptions such as topoisomerase-I or DNA-PK, inhibition of caspases is necessary to detect grB-mediated cleavage of the common targets (17
). Granule-mediated apoptosis is associated with grB-mediated cleavage of both ROCK proteins, and we show here that whereas ROCK I cleavage is indirect and requires the activation of caspases, grB cleaves ROCK II directly. This grB-mediated cleavage of ROCK II is very efficient in that it is readily detectable after 30 min of K562 cells being exposed to killer cells, in the absence or presence of caspase inhibitors. This observation is in accordance with our in vitro data showing that a minimal amount of grB is sufficient to process ROCK II.
Preventing caspase activation during granule-mediated apoptosis largely inhibits nuclear changes such as chromatin condensation or DNA oligonucleosomal fragmentation. In contrast, phosphatidylserine (PS) externalization, mitochondrial potential decrease and plasma membrane blebbing in target cells are not inhibited, suggesting the involvement of cytoplasmic caspase-independent events (11
). PS exposure could signal the phagocytosis of target cells despite incomplete nuclear apoptotic changes. Mitochondrial depolarization is likely caused by grB either as a consequence of Bid cleavage or via an alternative, Bid-independent pathway (18
). We show here that plasma membrane blebbing is another phenotypic feature of target cell apoptosis that can be directly mediated by grB through direct cleavage of ROCK II, thus ensuring the occurrence of this apoptotic change in cells with either defective or inhibited caspase activation.
Apoptotic bleb formation may have important functional consequences. Impaired clearance of apoptotic cells by macrophages is known to contribute to the development of autoimmune and inflammatory diseases (19
). Apoptotic membrane blebs express externalized PS and are a preferred site for complement C1q and C-reactive protein binding, events which are implicated in the noninflammatory clearance of dying cells (21
). Interestingly, most of the substrates cleaved by both caspase-3 and by grB are nuclear autoantigens that redistribute and cluster in blebs and/or apoptotic bodies (17
). Bleb recognition and engulfment by phagocytes permits the efficient removal of packaged caspase and/or grB substrates, and may participate in the maintenance of tolerance to self proteins.
The autoantigenic status of grB substrates, as opposed to the vast majority of caspase substrates, might be due to the exposure of cryptic and potentially more immunogenic epitopes that require swift uptake to avoid triggering of an autoimmune response (27
). During granule-mediated cell death, ROCK II cleavage by grB would maintain plasma membrane bleb formation and autoantigen clustering, even in the face of incomplete apoptotic signaling caused by deficient caspase activation in virus-infected or malignant target cells.