Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing

Abstract

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.

Molecular mechanisms of ATP secretion during immunogenic cell death

“…Thus, U2OS cells were treated with 1530 distinct chemicals from the ICCB Known Bioactives Library or the US-Drug Collection (encompassing most US Food and Drug Administration-approved and some experimental drugs), alone or in combination with MTX, for 18 h, stained with quinacrine and then analyzed by automated, quantitative epifluorescence microscopy ( Figure 2a). This screen led to the identification of several compounds that are capable of preventing the MTX-triggered loss of quinacrine-dependent fluorescence (Figure 2b): monensin, which blocks intracellular trafficking and exocytosis; 38 blebbistatin, which inhibits myosin II, thereby blocking the apoptosis-associated blebbing of the plasma membrane; 39 Y-27632, which inhibits ROCK1, 40 a protein that -upon cleavage by caspase-3 -participates in apoptotic blebbing; 41,42 and mefloquine, which inhibits PANX1 channels. 43 Subsequent validation experiments confirmed that monensin, blebbistatin and several chemically-unrelated inhibitors of ROCK1 (i.e., Y-27632, bearing with a central aminoethyl group, and H-1152, characterized by a central sulfonyl junction) and PANX1 (i.e., the synthetic quinine analogue mefloquine and the disulfonic stilbene derivatives 4,4 0 -diisothiocyano-2,2 0 -stilbenedisulfonic acid (DIDS) and 4-acetamido-4-isothiocyano-stilbene-2,2-disulfonic acid (SITS)) blunt the loss of quinacrine fluorescence as triggered in U2OS cells by MTX (Figure 2c).…”

Rho kinase in the regulation of cell death and survival

“…Increased MLC phosphorylation and actomyosin contraction in apoptotic cells regulate plasma membrane blebbing [88]. Caspase 3-mediated ROCK1 activation is responsible for the increased MLC phosphorylation in a variety of cell types [16,119]. Phosphatase and tensin homologue (PTEN) is a newly identified ROCK substrate [77].…”

“…ROCK1 is cleaved by caspase 3 at the cleavage site DETD1113 during apoptosis [16,119]. This consensus sequence for caspase 3 cleavage is conserved in human, rat and mouse, but is not present in ROCK2.…”

Redundant cell death mechanisms as relics and backups

“…The importance of this final stage especially in caspase-dependent apoptosis is emphasized by the existence of specialized pathways for dismantling cell structures, such as the pathways leading to DNA fragmentation 12 or to cell surface blebbing. 13 At least in the animal kingdom, this systematic and structure-specific dismantling, moreover followed by rapid engulfment of dying cells, 14,15 strongly suggests that there is a strict requirement for swift and complete disappearance of dead cells. Autophagy may ensure dismantling for some cell deaths both inside and outside of the animal kingdom, and in the latter kingdom recruitment of caspases as an extra tool for dismantling may have represented a further evolutionary advantage.…”