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Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.

Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. We report that Fas antigen is constitutively expressed in medullary epithelial cells of the human thymus. Expression is decreased in cultured thymic epithelial cells (TEC), similarly to HLA-DR antigen. TEC are resistant to anti-Fas-induced apoptosis after 4 days of primary culture, and this resistance is reversed by concomitant addition of cycloheximide. Cycloheximide also downregulated the expression of Fas-associated phosphatase-1, which has been found to inhibit Fas-induced apoptosis. This phosphatase could be involved in the resistance to Fas-induced apoptosis observed on day 4 of TEC culture. When TEC were subcultured after 10 to 13 days of primary culture, exposure to interleukin-1-beta, tumor necrosis factor-alpha, and interferon-gamma, alone or together, reinduced Fas mRNA and protein expression. In coculture with activated thymocytes, TEC also upregulated Fas protein expression. Cytokine-activated TEC became sensitive to apoptosis induced by an agonistic anti-Fas antibody. This apoptosis was inhibited by Z-VAD-fmk but not by Z-DEVD-fmk and DEVDase activity was slightly increased in Fas-stimulated TEC, suggesting that DEVDase activity is not sufficient to induce TEC apoptosis. Taken together, these data show that the Fas receptor is expressed in medullary epithelial cells of the human thymus and is able to induce apoptosis.