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

MAGI-1, a member of the MAGUK family of proteins, is shown to be rapidly cleaved during Fas-induced apoptosis in mouse 3T3 A31 cells, and in UV irradiation- and staurosporine-induced apoptosis in HaCaT cells. This generates a 97 kDa N-terminal fragment that dissociates from the cell membrane; a process that is largely prevented in the presence of the caspase inhibitor Z-VAD-fmk. In addition, we show that in vitro translated radiolabelled MAGI-1 is efficiently cleaved into 97 kDa and 68 kDa fragments by caspases-3 and -7 at physiological concentrations and mutating the MAGI-1 Asp₇₆₁ to Ala completely abolished the caspase-induced cleavage. Moreover, in HaCaT cells overexpressing the MAGI-1 Asp₇₆₁Ala mutant the disruption of cell-cell contacts was delayed during apoptosis, whereas other caspase-dependent processes such as nuclear condensation were not affected, suggesting that cell detachment is parallel to them. Thus, MAGI-1 cleavage appears to be an important step in the disassembly of cell-cell contacts during apoptosis.     

Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin

Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin.     

Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.     

The differentiation-dependent desmosomal cadherin desmoglein 1 is a novel caspase-3 target that regulates apoptosis in keratinocytes

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.     

Pro-apoptotic Role of Cdc25A: ACTIVATION OF CYCLIN B1/Cdc2 BY THE Cdc25A C-TERMINAL DOMAIN*

Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.     

Caspase proteolysis of the integrin beta4 subunit disrupts hemidesmosome assembly, promotes apoptosis, and inhibits cell migration

Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin beta4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its alpha6 partner, alpha6beta4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin beta4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp(1109)) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin beta4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1-1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin beta4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin beta4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.     

Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP)

The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.     

Linking sister chromatid cohesion and apoptosis: role of Rad21

Rad21 is one of the major cohesin subunits that holds sister chromatids together until anaphase, when proteolytic cleavage by separase, a caspase-like enzyme, allows chromosomal separation. We show that cleavage of human Rad21 (hRad21) also occurs during apoptosis induced by diverse stimuli. Induction of apoptosis in multiple human cell lines results in the early (4 h after insult) generation of 64- and 60-kDa carboxy-terminal hRad21 cleavage products. We biochemically mapped an apoptotic cleavage site at residue Asp-279 (D(279)) of hRad21. This apoptotic cleavage site is distinct from previously described mitotic cleavage sites. hRad21 is a nuclear protein; however, the cleaved 64-kDa carboxy-terminal product is translocated to the cytoplasm early in apoptosis before chromatin condensation and nuclear fragmentation. Overexpression of the 64-kDa cleavage product results in apoptosis in Molt4, MCF-7, and 293T cells, as determined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and Annexin V staining, assaying of caspase-3 activity, and examination of nuclear morphology. Given the role of hRad21 in chromosome cohesion, the cleaved C-terminal product and its translocation to the cytoplasm may act as a nuclear signal for apoptosis. In summary, we show that cleavage of a cohesion protein and translocation of the C-terminal cleavage product to the cytoplasm are early events in the apoptotic pathway and cause amplification of the cell death signal in a positive-feedback manner.     

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.     

Amplification of apoptosis through sequential caspase cleavage of the MET tyrosine kinase receptor

Activation of the MET tyrosine kinase receptor by hepatocyte growth factor/scatter factor is classically associated with cell survival. Nonetheless, stress stimuli can lead to a caspase-dependent cleavage of MET within its juxtamembrane region, which generate a proapoptotic 40 kDa fragment (p40 MET). We report here that p40 MET is in fact generated through an additional caspase cleavage of MET within its extreme C-terminal region, which removes only few amino acids. We evidenced a hierarchical organization of these cleavages, with the C-terminal cleavage favoring the juxtamembrane one. As a functional consequence, the removal of the last amino acids of p40 MET increases its apoptotic capacity. Finally, cells expressing a MET receptor mutated at the C-terminal caspase site are unable to generate p40 MET and are resistant to apoptosis, indicating that generation of p40 MET amplifies apoptosis. These results revealed a two-step caspase cleavage of MET resulting in the reshaping of this survival receptor to a proapoptotic factor.     

Cleavage of the Junin Virus Nucleoprotein Serves a Decoy Function To Inhibit the Induction of Apoptosis during Infection

The regulation of apoptosis during infection is an important factor for host survival and, in some cases, also for the virus life cycle. At the same time, mechanisms to prevent the induction of apoptosis have been observed in numerous viral pathogens, but until now the role of apoptosis during arenavirus infection has not been investigated. Junin virus (JUNV) belongs to the New World arenavirus serogroup of the Arenaviridae and is the causative agent of Argentine hemorrhagic fever. We have demonstrated that infection with JUNV in cell culture does not induce apoptosis but leads to cleavage of the nucleoprotein (NP) into discrete products resembling caspase cleavage events. Similar specific NP degradation patterns were also observed in NP-transfected cell lines, and a closer examination of the sequence of NP showed several putative caspase cleavage motifs. Point mutations that abolished these cleavage motifs were consistent with the loss of certain cleavage products. Consistent with these data, further studies showed that treatment with a caspase inhibitor also reduced NP cleavage, indicating that the observed cleavage events were occurring as a result of caspase activity with NP as a substrate. Finally, we showed that expression of NP suppresses the cleavage of caspase 3 in cells treated with an apoptosis activator. Based on these findings, we propose that NP functions as a decoy substrate for caspase cleavage in order to inhibit the induction of apoptosis in JUNV-infected cells.     

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.