Caspase-mediated cleavage of JNK during stress-induced apoptosis

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Caspase-mediated cleavage of the TIAM1 guanine nucleotide exchange factor during apoptosis.

Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH(2)-terminal of TIAM's two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH(2)-terminal kinase and serum response factor. Finally, we demonstrate that in cells treated with ceramide, cleavage of TIAM1 coincided with the inactivation of endogenous Rac. These results reveal a novel mechanism for regulating guanine nucleotide exchange factor activity and GTPase-mediated signaling pathways.     

Caspase-mediated cleavage of the Ca2+/calmodulin-dependent protein kinase-like kinase facilitates neuronal apoptosis

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.     

Proteolytic cleavage of ras GTPase-activating protein during apoptosis

p120-ras GTPase-activating protein (rasGAP) associates with Ras and negatively regulates Ras signaling by stimulating the intrinsic rate of Ras GTPase activity. rasGAP also associates with other cellular signaling proteins which suggest that rasGAP may play a role in coordinating other signal transduction pathways. Disruption of rasGAP in vivo results in extensive apoptosis. Fas-mediated apoptosis results in the activation of caspases that cleave cellular substrates which are important for maintaining cytoplasmic and nuclear integrity. We show here that rasGAP is proteolytically cleaved by caspases early in Fas-induced apoptosis of Jurkat cells. rasGAP was also cleaved by DNA-damaging chemotherapeutic agents and TNF-related apoptosis inducing ligand (TRAIL), also known as Apo2L. Based on the size of the products generated by cleavage of deletion mutants of rasGAP we predict that cleavage of rasGAP occurs in the hydrophobic region and between the SH2(2) and ras-p21 interacting domain which would leave an intact ras-p21 interacting domain. Interestingly, cleavage of rasGAP in vitro enhanced rasGAP hydrolysis activity. Our results demonstrate that diverse apoptotic stimuli cause caspase-mediated cleavage of rasGAP early in apoptosis.     

Caspase-mediated changes in Sir2alpha during apoptosis

Silent information regulator 2 (Sir2) is an NAD(+)-dependent histone deacetylase that establishes repressive chromatin status and extends the life span of both budding yeast and the nematode worm Caenorhabditis elegans. There is growing evidence that its mammalian homologue Sir2alpha protects cells from stress-induced apoptosis. We report here that mammalian Sir2alpha was directly cleaved by both initiator and executioner caspases, and relocated from the nucleus to the cytoplasm in apoptotic cells. These alterations of Sir2alpha were largely inhibited by a caspase-9 dominant-negative mutant or Bcl-xL. Our results indicate that Sir2alpha undergoes dynamic changes in caspase-dependent manner during apoptosis.     

Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp32/PHAP-I regulation of apoptosis

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.     

Caspase-mediated cleavage of actin-binding and SH3-domain-containing proteins cortactin, HS1, and HIP-55 during apoptosis.

Reorganization of the actin cytoskeleton occurs during apoptosis. We found that actin-binding and Src homology 3 (SH3)-domain-containing proteins cortactin, hematopoietic-specific protein 1 (HS1), and hematopoietic progenitor kinase 1-interacting protein of 55 kDa (HIP-55, also called SH3P7 and Abp1) were degraded in a caspase-dependent manner during apoptosis. Cortactin, HS1, and HIP-55 were direct substrates of caspase 3. Cortactin and HS1 have two clusters of potential caspase cleavage sites; one is in their actin-binding domains, and the other is close to their carboxy-terminal SH3 domains. HIP-55 has one caspase recognition site, EHID(361). The HIP-55 (D361A) mutant was resistant to caspase cleavage. Cleavage of HIP-55 by caspases dissociated its actin-binding domain from its SH3 domain. The cleavage of these actin-binding and SH3 domain-containing proteins may affect cell signaling to and from the actin cytoskeleton and may be involved in the morphological change of cells during apoptosis.     

Caspase-mediated parkin cleavage in apoptotic cell death

The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.     

Caspase-mediated specific cleavage of human histone deacetylase 4

Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation.     

cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope.

Anti-Sm antibodies from a patient with systemic lupus erythematosus (SLE) were used to isolate cDNA clones encoding the snRNP-associated protein N from a rat brain derived cDNA library. The predicted primary structure of the 240 amino acid protein has a proline rich carboxyl terminus and shares a region of sequence similarity with other snRNP polypeptides, A and B/B'. Anti-Sm sera recognize a beta-galactosidase fusion protein containing only the carboxyl-terminal 80 amino acids of N; antibodies eluted from this fusion protein also react with A, B/B' and N on immunoblots, suggesting that these proteins share an Sm epitope located within this segment. Polyclonal antibodies raised against a 23 amino acid synthetic peptide derived from this conserved region of N recognize A, N and B/B' on immunoblots and can immunoprecipitate the Sm class of U snRNAs. These results confirm that this sequence defines a potential Sm epitope. RNA blotting analyses demonstrate that a 1.6 kb mRNA expressed predominantly in brain encodes the N polypeptide in both rats and humans. At low stringency rat N cDNA also hybridizes to a 1.3 kb mRNA species which encodes B/B', suggesting that N is structurally related to, but distinct from B/B'. Although B/B' proteins are thought to be expressed in all human cells, only N and B, but not B', are observed on immunoblots of human brain proteins probed with anti-Sm sera. The apparent difference in the complement of proteins associated with snRNP particles in human brain versus elsewhere suggests a possible mechanism for the regulation of brain-specific mRNA splicing.     

Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.     

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.     

Proteolytic cleavage during chemotherapy-induced apoptosis

Treatment with anticancer drugs sets into motion a morphologically and biochemically distinct type of cell death called apoptosis. Recent genetic and biochemical studies have suggested that proteases play a prominent role in the active phase of apoptotic cell death. Ongoing studies are aimed at identifying the proteases involved, the substrates that are cleaved, and the means by which the proteolytic process is regulated in nonapoptotic and apoptotic cells. The possibility that these findings will suggest new approaches to treating cancer and other diseases is discussed.     

The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase.

SR proteins play important roles in the recognition and selection of the 3' and 5' splice site of a given intron and contribute to the phosphorylation/dephosphorylation-mediated regulation of pre-mRNA splicing. Recent studies have demonstrated that the U1 snRNP is recruited to the 5' splice site by protein/protein interactions involving the SR domains of the U1-70K protein and SF2/ASF. Recently, it was suggested that SR proteins might also contribute to the binding of the [U4/U6.U5] tri-snRNP to the pre-spliceosome (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706), although it remains unclear whether these SR proteins interact with proteins of the tri-snRNP complex. As a first step toward the identification of proteins that could potentially mediate the integration of the [U4/U6.U5] tri-snRNP complex into the spliceosome, we investigated whether purified [U4/U6.U5] tri-snRNP complexes contain SR proteins. Three proteins in the tri-snRNP complex with approximate molecular weights of 27, 60, and 100 kDa were phosphorylated by purified snRNP-associated protein kinase, which has been shown previously to phosphorylate the serine/ arginine-rich domains of U1-70K and SF2/ASF (Woppmann A et al., 1993, Nucleic Acids Res 21:2815-2822). These proteins are thus prime candidates for novel tri-snRNP SR proteins. Here, we describe the biochemical and molecular characterization of the 27K protein. Analysis of a cDNA encoding the 27K protein revealed an N-terminal SR domain strongly homologous (54% identity) to the SR domain of the U1 snRNP-specific 70K protein. In contrast to many other SR proteins, the 27K protein does not contain an RNA-binding domain. The 27K protein can be phosphorylated in vitro by the snRNP-associated protein kinase and exhibits several isoelectric variants upon 2D gel electrophoresis. Thus, the tri-snRNP-specific 27K protein could potentially be involved in SR protein-mediated protein/protein interactions and, additionally, its phosphorylation state could modulate pre-mRNA splicing.     

Caspase-mediated cleavage of X-ray repair cross-complementing group 4 promotes apoptosis by enhancing nuclear translocation of caspase-activated DNase

X-ray repair cross-complementing group 4 (XRCC4), a repair protein for DNA double-strand breaks, is cleaved by caspases during apoptosis. In this study, we examined the role of XRCC4 in apoptosis. Cell lines, derived from XRCC4-deficient M10 mouse lymphoma cells and stably expressing wild-type XRCC4 or caspase-resistant XRCC4, were established and treated with staurosporine (STS) to induce apoptosis. In STS-induced apoptosis, expression of wild-type, but not caspase-resistant, XRCC4 in XRCC4-deficient cells enhanced oligonucleosomal DNA fragmentation and the appearance of TUNEL-positive cells by promoting nuclear translocation of caspase-activated DNase (CAD), a major nuclease for oligonucleosomal DNA fragmentation. CAD activity is reportedly regulated by the ratio of two inhibitor of CAD (ICAD) splice variants, ICAD-L and ICAD-S mRNA, which, respectively, produce proteins with and without the ability to transport CAD into the nucleus. The XRCC4-dependent promotion of nuclear import of CAD in STS-treated cells was associated with reduction of ICAD-S mRNA and protein, and enhancement of phosphorylation and nuclear import of serine/arginine-rich splicing factor (SRSF) 1. These XRCC4-dependent, apoptosis-enhancing effects were canceled by depletion of SRSF1 or SR protein kinase (SRPK) 1. In addition, overexpression of SRSF1 in XRCC4-deficient cells restored the normal level of apoptosis, suggesting that SRSF1 functions downstream of XRCC4 in activating CAD. This XRCC4-dependent, SRPK1/SRSF1-mediated regulatory mechanism was conserved in apoptosis in Jurkat human leukemia cells triggered by STS, and by two widely used anti-cancer agents, Paclitaxel and Vincristine. These data imply that the level of XRCC4 expression could be used to predict the effects of apoptosis-inducing drugs in cancer treatment.     

Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr⁷⁰⁵) and of tyrosine phosphatase SHP2 (Tyr⁵⁸⁰). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18 kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.     

Stimuli-dependent cleavage of Dicer during apoptosis

The 'stalk' is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.