Proteolytic cleavage of phospholipase C-gamma1 during apoptosis in Molt-4 cells.

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospholipase C-gamma1 (PLC-gamma1), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH(3) potentiated etoposide-induced apoptosis in these cells. PLC-gamma1 was fragmented when Molt-4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor alpha. Cleavage of PLC-gamma1 was blocked by overexpression of Bcl-2 and by specific inhibitors of caspases such as Z-DEVD-CH(2)F and YVAD-cmk. Purified caspase-3 and caspase-7, group II caspases, cleaved PLC-gamma1 in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC-gamma1 is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala-Glu-Pro-Asp(770) was identified to be a cleavage site within PLC-gamma1. Epidermal growth factor receptor (EGFR) -induced tyrosine phosphorylation of PLC-gamma1 resulted in resistance to cleavage by caspase-3 in vitro. Furthermore, cleaved PLC-gamma1 could not be tyrosine-phosphorylated by EGFR in vitro. In addition, tyrosine-phosphorylated PLC-gamma1 was not significantly cleaved during etoposide-induced apoptosis in Molt-4 cells. This suggests that the growth factor-induced tyrosine phosphorylation may suppress apoptosis-induced fragmentation of PLC-gamma1. We provide evidence for the biochemical relationship between PLC-gamma1-mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC-gamma1-mediated signaling pathway can facilitate an apoptotic progression.     

The fate of desmosomal proteins in apoptotic cells

Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.     

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.     

The N-terminal fragment from caspase-cleaved serine/arginine protein-specific kinase2 (SRPK2) translocates into the nucleus and promotes apoptosis

SRPK2 belongs to a family of serine/arginine (SR) protein-specific kinases (SRPKs), which phosphorylate SR domain-containing proteins in the nuclear speckles and mediate the pre-mRNA splicing. Previous studies have shown that SRPK2 plays a pivotal role in cell proliferation and apoptosis. However, how SRPK2 is regulated during the apoptosis is unclear. Here, we show that SRPK2 is cleaved by caspases at Asp-139 and -403 residues. Its N terminus cleaved product translocates into the nucleus and promotes VP16-induced apoptosis. Akt phosphorylation of SRPK2 prevents its apoptotic cleavage by caspases. 14-3-3β, the binding partner of Akt-phosphorylated SRPK2, further protects it from degradation. Hence, our results suggest that the N-terminal domain of SRPK2 cleaved by caspases translocates into the nucleus, where it promotes chromatin condensation and apoptotic cell death.     

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.     

Bcl-2 cleavages at two adjacent sites by different caspases promote cisplatin-induced apoptosis

The protein encoded by bcl-2 proto-oncogene plays an important role in the mitochondria-mediated apoptotic pathway. Although the general role of Bcl-2 is anti-apoptotic, previous work showed that Bcl-2 fragments cleaved by caspases could promote apoptotic process. We report herein that Bcl-2 protein was cleaved to produce two fragments of around 23 kDa in human hepatocarcinoma BEL-7404 cells or in Bcl-2 overexpressing CHO cells induced by cisplatin. Treating cells with the general caspase inhibitor z-VAD-fmk blocked the induced cleavage of Bcl-2. Mutagenesis analyses showed that Bcl-2 was cleaved by caspases at two adjacent recognition sites in the loop domain （YEWD31↓AGD34↓V）, which could be inhibited by caspase-8 and -3 inhibitors, respectively. Overexpression of the carboxyl terminal 23 kDa fragments increased the sensitivity of CHO cells to cisplatin-induced apoptosis. These results indicate that Bcl-2 can be cleaved into two close fragments by different caspases during cisplatin-induced apoptosis, both of which contribute to the acceleration ofapoptotic process.     

Caspase-dependent cleavage disrupts the ERK cascade scaffolding function of KSR1

Kinase suppressor of Ras 1 (KSR1) is a protein scaffold that facilitates ERK cascade activation at the plasma membrane, a critical step in the signal transduction process that allows cells to respond to survival, proliferative, and differentiative cues. Here, we report that KSR1 undergoes caspase-dependent cleavage in apoptotic cells and that cleavage destroys the scaffolding function of the full-length KSR1 protein and generates a stable C-terminal fragment that can inhibit ERK activation. KSR1 is cleaved in response to multiple apoptotic stimuli and occurs in vivo during the involution of mouse mammary tissues, a morphogenic process requiring cellular apoptosis. In addition, we find that in comparison with KSR1(-/-) mouse embryonic fibroblasts expressing wild type KSR1 (WT-KSR1), cells expressing a cleavage-resistant KSR1 protein (DEVA-KSR1) exhibit reduced apoptotic signaling in response to tumor necrosis factor-alpha/cycloheximide treatment. The effect of DEVA-KSR1 expression was found to correlate with increased levels of active phosphoERK and could be significantly reversed by treating cells with the MEK inhibitor U0126. In contrast, reduced phosphoERK levels and enhanced apoptotic signaling were observed in cells constitutively expressing the C-terminal KSR1 fragment (CTF-KSR1). Moreover, we find that cleavage of WT-KSR1 correlates with a dramatic reduction in active phosphoERK levels. These findings identify KSR1 as a caspase target and suggest that cleavage of the KSR1 scaffold represents another mechanism whereby caspases down-regulate ERK survival signaling to promote cellular apoptosis.     

Cleavage and inactivation of antiapoptotic Akt/PKB by caspases during apoptosis

The oncogene Akt/PKB/RAC-PK is a serine/threonine kinase that mediates survival signals and has protective effects against apoptosis induced by a variety of stimuli. The kinase activity of Akt has been demonstrated to be critical in transmitting survival signals. We found that Akt protein was down-regulated during apoptosis. The down-regulation was blocked by a caspase inhibitor, indicating that Akt was cleaved by caspases during apoptosis. The Akt protein incubation with active caspases in vitro revealed that it was cleaved at three sites to produce 40- and 44-kDa fragments. The two cleavage sites were between the NH(2)-terminal pleckstrin homology domain (PH domain) and the kinase domain (TVAD(108 downward arrow)G and EEMD(119 downward arrow)F) and in the COOH-terminal regulatory domain (SETD(434 downward arrow)T). The loss of COOH-terminal domain of the Akt protein reduced its kinase activity and the overexpression of NH(2)-terminal and COOH-terminal-deleted Akt fragment increased the sensitivity to apoptosis-inducing stimuli. These results indicate that caspase-dependent cleavage of anti-apoptotic Akt turns off the survival signals, resulting in the acceleration of apoptotic cell death.     

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.     

TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects.     

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.     

Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.

Dok (for downstream of tyrosine kinases) proteins are a newly identified family of docking molecules that are characterized by the presence of an amino-terminal pleckstrin homology (PH) domain, a central putative phosphotyrosine-binding (PTB) domain and numerous potential sites of tyrosine phosphorylation [1] [2] [3] [4] [5] [6]. Here, we explore the potential role of the Dok family member Dok-R (also known as p56(Dok2) or FRIP) in signaling pathways mediated by the epidermal growth factor (EGF) receptor. An intact PTB domain in Dok-R was critical for its association with two PTB-binding consensus sites on the EGF receptor and the PH domain further contributed to stable in vivo binding and tyrosine phosphorylation of Dok-R. Multiple sites on Dok-R were tyrosine-phosphorylated following EGF stimulation; phosphorylated Tyr276 and Tyr304 are proposed to dock the tandem Src homology 2 (SH2) domains of the p21(Ras) GTPase-activating protein rasGAP and Tyr351 mediates an association with the SH2 domain of the adapter protein Nck. Interestingly, we have found that Dok-R could attenuate EGF-stimulated mitogen-activated protein (MAP) kinase activation independently of its association with rasGAP. Together, these results suggest that Dok-R has an important role downstream of growth factor receptors as a potential negative regulator of signal transduction.     

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Cellular localization of MAGI-1 caspase cleavage products and their role in apoptosis

MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein present at adherent and tight junctions, where it acts as a structural and signaling scaffold. During apoptosis, MAGI-1 is cleaved by caspases at Asp761 into N- and C-terminal cleavage products, allowing further dismantling of the cell junctions, one of the key features of apoptosis. Here, we investigated the cellular distribution and possible proapototic role of MAGI-1 caspase cleavage products. Full-length MAGI-1 exhibited submembrane localization, while the N-terminal caspase cleavage product of MAGI-1 is translocated to the cytosol and the C-terminal caspase cleavage product accumulates in the nucleus. When overexpressed in MDCK cells, both N- and C-terminal MAGI-1 caspase cleavage products exhibited minor proapoptotic activity, although their role in apoptosis is probably more passive.     

Cleavage of calnexin caused by apoptotic stimuli: implication for the regulation of apoptosis

Calnexin is an endoplasmic reticulum (ER)-resident molecular chaperone that plays an essential role in the correct folding of membrane proteins. We found that calnexin is subjected to partial cleavage in apoptotic mouse cells. Both ER stress-inducing and ER stress-non-inducing apoptotic stimuli caused the cleavage of calnexin, indicating that this event does not always occur downstream of ER stress. The inhibition of caspases that target the amino acid sequence DXXD abrogated calnexin cleavage in apoptotic stimulus-treated cells. In addition, disruption of one of two DXXD sequences located in the cytoplasmic domain caused calnexin to escape cleavage during apoptosis. Furthermore, calnexin was cleaved in vitro by recombinant caspase-3 or caspase-7. Finally, the overexpression of a presumed cleavage product of calnexin partly inhibited apoptosis. These results collectively suggest that caspase-3 or caspase-7 cleaves calnexin, whose cleaved product leads to the attenuation of apoptosis.     

Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.     

Caspase cleavage of HER-2 releases a Bad-like cell death effector

Human epidermal growth factor receptor-2 (HER-2/ErbB2/neu), a receptor tyrosine kinase that is amplified/overexpressed in poor prognosis breast carcinomas, confers resistance to apoptosis by activating cell survival pathways. Here we demonstrate that the cytoplasmic tail of HER-2 is cleaved by caspases at Asp(1016)/Asp(1019) to release a approximately 47-kDa product, which is subsequently proteolyzed by caspases at Asp(1125) into an unstable 22-kDa fragment that is degraded by the proteasome and a predicted 25-kDa product. Both the 47- and 25-kDa products translocate to mitochondria, release cytochrome c by a Bcl-x(L)-suppressible mechanism, and induce caspase-dependent apoptosis. The 47- and 25-kDa HER-2 cleavage products share a functional BH3-like domain, which is required for cytochrome c release in cells and isolated mitochondria and for apoptosis induction. Caspase-cleaved HER-2 binds Bcl-x(L) and acts synergistically with truncated Bid to induce apoptosis, mimicking the actions of the BH3-only protein Bad. Moreover, the HER-2 cleavage products cooperate with Noxa to induce apoptosis in cells expressing both Bcl-x(L) and Mcl-1, confirming their Bad-like function. Collectively, our results indicate that caspases activate a previously unrecognized proapoptotic function of HER-2 by releasing a Bad-like cell death effector.