A subset of caspase substrates functions as the Jekyll and Hyde of apoptosis

Cleavage of caspase substrates is believed to be the commitment point that will lead a cell towards apoptosis. While the cleavage of some caspase substrates participates directly in the dismantling of the cell, others regulate the extent of caspase activation. In this communication, we discuss some recent findings indicating that two caspase substrates, MEKK1 and RasGAP, change their functions from anti- to pro-apoptotic as caspase activity increases. MEKK1 is a MAPK kinase kinase regulating the JNK MAPK pathway. As a full-length protein, MEKK1 generates protective signals (e.g. in cardiomyocytes), but potentiates apoptosis when cleaved by caspases. This switch is mediated by a translocation of the kinase activity from insoluble to soluble cellular structures. RasGAP is a regulator of Ras GTPase family members. As a full-length protein, RasGAP does not modulate apoptosis. However, low caspase activity readily induces the cleavage of RasGAP into an N-terminal fragment that generates potent anti-apoptotic signals. At higher caspase activity, the N-terminal fragment is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP can, thus, be viewed as an apoptostat because it allows the cells to determine when caspases have been mildly activated to fulfill functions other than apoptosis or when caspases are strongly activated to mediate apoptosis.     

Impaired Akt activity down-modulation, caspase-3 activation, and apoptosis in cells expressing a caspase-resistant mutant of RasGAP at position 157

RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apoptosis. Therefore, the role of the second caspase-mediated cleavage of RasGAP is to allow the inactivation of the antiapoptotic function of fragment N so that caspases are no longer hampered in their ability to kill cells.     

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart.     

The RasGAP N-terminal fragment generated by caspase cleavage protects cells in a Ras/PI3K/Akt-dependent manner that does not rely on NFkappa B activation

RasGAP, a regulator of Ras GTPase family members, is cleaved at low levels of caspase activity into an N-terminal fragment (fragment N) that generates potent anti-apoptotic signals. At higher levels of caspase activity, fragment N is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP could thus function as a sensor of caspase activity to determine whether a cell should survive or not. Here we show that fragment N protects cells by activating the Ras-PI3K-Akt pathway. Surprisingly, even though nuclear factor kappaB (NFkappaB) can be activated by Akt, it plays no role in the anti-apoptotic functions of fragment N. This indicates that Akt effectors are differentially regulated when fragment N is generated.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.     

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.     

Anamorsin,a Novel Caspase-3 Substrate in Neurodegeneration

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD²⁰⁹↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.     

SfDronc,an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.     

Many cuts to ruin: a comprehensive update of caspase substrates

Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.     

Caspase Activation Accompanying Cytochrome c Release from Mitochondria Is Possibly Involved in Nitric Oxide-Induced Neuronal Apoptosis in SH-SY5Y Cells

Abstract : It is well known that caspases are produced as proforms, which are proteolytically cleaved and activated during apoptosis or programmed cell death. We report here that caspases are activated during apoptosis by treatment with NOC18, a nitric oxide (NO) donor. Our present experiments have examined the way in which NO induces neuronal cell death, using a new type of NO donor that spontaneously releases only NO without enzymatic metabolism. NOC18 induced apoptosis in human neuroblastoma SH-SY5Y cells in a concentration-and time-dependent manner as estimated by DNA fragmentation assay, FACScan analysis, and nuclear morphology. Oxyhemoglobin, an NO trapper, suppressed NOC18-triggered DNA fragmentation, indicating that NO from NOC18 is a real activator in this study. Upon the induction of apoptosis, an increase in caspase-3-like protease activity, but not caspase-1, was observed. Procaspase-2 protein, an inactive form of caspase-2, decreased dramatically. In addition, NOC18 also resulted in poly (ADP-ribose) polymerase (PARP) cleavage, yielding an 85-kDa fragment typical of caspase activity. Oxyhemoglobin blocked the decrease of procaspase-2 and the cleavage of PARP by NOC18 in a concentration-dependent manner. Moreover, NO elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that both stimulation of caspase activity and cytochrome c release are partly involved in NO-induced neuronal apoptosis.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH₂-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.