Specific Cleavage of α-Fodrin during Fas- and Tumor Necrosis Factor-induced Apoptosis Is Mediated by an Interleukin-1β-converting Enzyme/Ced-3 Protease Distinct from the Poly(ADP-ribose) Polymerase Protease

Abstract

Interleukin-1␤-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that ␣-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas-and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/ Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-ValAsp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.

Caspases: the executioners of apoptosis

“…α-Fodrin, an abundant membrane-associated cytoskeletal protein, is rapidly and specifically cleaved during CD95-and TNF-induced apoptosis, and this appears to be related to the membrane blebbing. Initially it was proposed that fodrin is cleaved by calpain I [127], but the cleavage is probably due to a caspase [128,129]. Fodrin contains several potential caspase cleavage sites, including a DETD S site only nine amino acids away from the proposed calpain cleavage site, which may have led to the initial confusion [128].…”

“…Cleavage of important cytoskeletal proteins, including actin [124,125], Gas2 [126] and fodrin (non-erythroid spectrin) [127][128][129], during apoptosis may induce cell shrinkage and membrane blebbing, and alter cell survival signalling systems. α-Fodrin, an abundant membrane-associated cytoskeletal protein, is rapidly and specifically cleaved during CD95-and TNF-induced apoptosis, and this appears to be related to the membrane blebbing.…”

Cleavage of MAGI-1, a tight junction PDZ protein, by caspases is an important step for cell-cell detachment in apoptosis

“…Several of them have already been shown to be cleaved by caspases, among them: β-catenin, plakoglobin [6][7][8], α-fodrin [9], actin [10], gelsolin [11], E-cadherin [12], focal adhesion kinase [13], desmosomal plaque proteins [14], the tight junction proteins ZO-1, ZO-2 and occludin, [15] and also hDLG, a constituent of adherens junctions in epithelial and endothelial cells [16].…”

Analysis of mechanisms involved in the prevention of γ irradiation-induced apoptosis by hGM-CSF

“…Caspase-1-like enzyme activity was not observed under the same conditions (data not shown). Among several putative caspases cellular substrate proteins, a-fodrin is one reported to be degraded by caspase from 240 to 150 kDa and 120 kDa fragments (Cryns et al, 1996). We next examined degradation of a-fodrin after factor depletion from BA/F3 cells by Western blotting of the total cell lysate.…”