Predominant Identification of RNA-binding Proteins in Fas-induced Apoptosis by Proteome Analysis*

Bernd Thiede, Christiane Dimmler, Frank Siejak, and Thomas RudelDagger

From the Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstr. 21/22, D-10117 Berlin, Germany

Received for publication, February 5, 2001, and in revised form, April 26, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54nrb, SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha  NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptosis, a genetically determined form of cellular suicide, is an essential and complex process involved in the development and maintenance of cell homeostasis in multicellular organisms. Improper regulation of apoptosis has been connected with various diseases including cancer, autoimmune disorders, viral infections, AIDS, neurodegenerative disorders, and myocardial infarction (1). Because of its extraordinary importance in human diseases, the therapeutic regulation of apoptosis offers numerous challenges (2).

The basis for therapeutic intervention, however, is the identification of the molecular players involved in apoptosis regulation. During the last few years, numerous factors constituting the machinery that initiates and regulates apoptosis have already been characterized. Specialized receptors, so-called death receptors, of the tumor necrosis factor superfamily induce apoptosis by ligand binding and receptor oligomerization, followed by the recruitment of adapter and effector proteins to the death domain of the receptor (3). One of the best characterized death receptor is the so-called CD95 (Fas/Apo-1) receptor that plays a major role in controlling immune cells. CD95(Fas/Apo-1) is involved in the deletion of autoreactive cells and activation-induced T cell death, killing of virus-infected cells or cancer cells, and killing of inflammatory cells at immune privileged sites (4-6).

The effector molecules activated by death receptors are the caspases (7, 8), a family of cysteinyl-aspartases that execute the apoptotic program by cleaving different cellular substrates (9). Cleavage of the substrate results in the destruction of cytoskeletal proteins (10, 11), activation or inactivation of signaling molecules (12, 13), and conversion of inhibitors into activators of apoptosis (14). The net effect of substrate processing is the ordered destruction of the cell body including fragmentation of DNA and formation of fragmented cell particles, apoptotic bodies, which are engulfed by neighboring cells (15). To understand the molecular details of the destruction process, the identification of caspase substrates involved in the elementary processes of apoptosis is essential.

Proteome approaches have been successfully used to find new apoptosis-associated proteins (16, 17). The proteome was defined as the protein complement expressed by the genome of an organism, tissue, or differentiated cell (18). The study of proteomes is one of the most important approaches to understand gene function (19, 20). Medical and clinical applications of proteomics to diseases promise to identify new diagnostic markers and aid in the development of drugs (21, 22). The proteins are separated by two-dimensional gel electrophoresis for proteome analysis (23, 24). The subtractive analysis of the two-dimensional gel electrophoresis pattern of a control with the process of interest; e.g. a specific disease can lead to different protein spots (25). These spots can be identified by various methods. Electrospray ionization mass spectrometry (26) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)1 (27) are particularly suitable for this purpose due to their sensitivity, speed, and accuracy (28). Finally, the obtained proteome should be stored in a data base to handle the large amount of information (29-31).

We recently established an Internet-accessible two-dimensional gel electrophoresis data base (available on the World Wide Web) of the Jurkat T cells (32). In order to identify proteins that are modified during apoptosis, we used the two-dimensional gel electrophoresis data base as a reference to investigate the proteome of Jurkat T cells that had been induced to undergo apoptosis. 37 apoptosis-modified spots of 21 different proteins were identified. Interestingly, 15 of these proteins contain an RNA-binding motif (33), and 12 are involved in the splicing process.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- The Jurkat T cell line E6 was maintained in RPMI tissue culture medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies) and penicillin (100 units/ml)/streptomycin (100 µg/ml) (Life Technologies) at 37 °C in 5.0% CO2.

Induction of Apoptosis-- Apoptosis was induced in 2 × 106 Jurkat T cells for 6 h at 37 °C in 5.0% CO2 by 250 ng/ml alpha CD95 (clone CH11) (Immunotech, Marseille, France). 1 µg/ml cycloheximide was added to the control- and the Fas-induced cells.

Separation of the Compartments-- Approximately 1 × 108 Jurkat T cells were centrifuged for 10 min at 1300 units/min at room temperature in a Megafuge 1.0R (Heraeus Instruments GmbH, Hanau, Germany). The supernatant was discarded, and the pellet was washed twice with 10 ml of phosphate-buffered saline (Life Technologies) and once with MB buffer (400 mM sucrose, 50 mM Tris, 1 mM EGTA, 5 mM 2-mercaptoethanol, 10 mM potassium hydrogen phosphate, pH 7.6, and 0.2% BSA) and centrifuged as above. The pellet was suspended in MB buffer (4 ml/108 cells) and incubated on ice for 20 min. Subsequently, the cells were homogenized and centrifuged at 3500 units/min for 1 min at 4 °C (Rotor SS-34; Sorvall RC5B, Hanau, Germany). The supernatant contained the mitochondria/cytosol/membranes, and the pellet enclosed the nucleus.

The mitochondrial fraction was pelleted by centrifugation at 8600 units/min for 10 min at 4 °C (Rotor SS-34; Sorvall RC5B). The supernatant contained the cytosol and membranes.

The pellet was suspended in MSM buffer (10 mM potassium hydrogen phosphate, pH 7.2, 0.3 mM mannitol, and 0.1% BSA) (0.4 ml/108 cells) and purified by sucrose gradient centrifugation in 10 ml of SA buffer (1.6 M sucrose, 10 mM potassium hydrogen phosphate, pH 7.5, and 0.1% BSA) and 10 ml of SB buffer (1.2 M sucrose, 10 mM potassium hydrogen phosphate, pH 7.5, and 0.1% BSA) at 20,000 units/min for 1 h at 4 °C (Rotor SW-28; Beckman L8-70M Ultracentrifuge, München, Germany). The interphase that contained the mitochondria was collected, suspended in 4 volumes of MSM buffer, and centrifuged again at 15,500 units/min for 10 min at 4 °C (Rotor SS-34; Sorvall RC5B). The pellet was suspended in MSM buffer without BSA and could be stored at -70 °C.

The supernatant with the cytosol and membrane was centrifuged at 100,000 units/min for 20 min at 4 °C (Rotor TLA120.2 rotor, Ultracentrifuge Optima TLX, Beckman, München, Germany). The pellet contained the membrane.

The pellet with the nucleus was suspended in 5 ml of phosphate-buffered saline and centrifuged for 2 min at 3500 units/min at 4 °C (Rotor SS-34; Sorvall RC5B). The pellet was suspended in NB buffer (10 mM Hepes, pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, and 1 mM pefabloc) (1 ml/108 cells) and incubated for 1 h on ice, subsequently homogenized, and applied to 10 ml of 30% sucrose in NB buffer. After the centrifugation with the Megafuge 1.0R (Heraeus Instruments) at 2000 units/min for 10 min at 4 °C, the pellet was washed twice with 6 ml of NB buffer, centrifuged as above, suspended in 1 ml of NB buffer, and centrifuged again at 10,000 units/min for 10 min at 4 °C (Rotor SS-34; Sorvall RC5B). The pellet could be stored at -70 °C.

Two-dimensional Gel Electrophoresis-- The proteins were separated by a large gel two-dimensional gel electrophoresis technique (gel size was 30 × 23 cm) and stained as previously described (32). Briefly, isoelectric focusing rod gels were used for the first dimension with a diameter of 0.9 mm for analytical gels and 2.5 mm for preparative gels. SDS-polyacrylamide gels with 15% (w/v) acrylamide and 0.2% bisacrylamide were used for the second dimension (34). Preparative gels were stained with Coomassie Brilliant Blue R-250 or G-250 (Serva, Heidelberg, Germany). Analytical gels were stained with silver nitrate (35;36).

Tryptic Digestion-- The Coomassie Blue-stained single gel spots from Jurkat T cells were excised with a scalpel for in-gel digestion with 0.1 µg of trypsin (Promega, Madison, WI) in 20 µl of 50 mM ammonium bicarbonate, pH 7.8. The samples were dissolved in 1 µl of 0.5% aqueous trifluoroacetic acid/acetonitrile (2:1) for the mass spectrometric analysis.

Peptide Mass Fingerprinting by MALDI-MS-- The mass spectra were recorded by using a time-of-flight delayed extraction MALDI mass spectrometer (Voyager-Elite, Perseptive Biosystems, Framingham, MA). 20 mg/ml alpha -cyano-4-hydroxycinnamic acid in 0.3% aqueous trifluoroacetic acid/acetonitrile (1:1) or 50 mg/ml 2,5-dihydroxybenzoic acid in 0.3% aqueous trifluoroacetic acid/acetonitrile (2:1) was used as matrix. The samples were applied to a gold-plated sample holder and introduced into the mass spectrometer after drying. The spectra were obtained in the reflectron mode by summing 70-200 laser shots.

Data Base Searching-- The proteins were identified by using the peptide mass fingerprinting analysis software MS-Fit (available on the World Wide Web). The NCBI and SwissProt data bases with the species human and mouse were used for the searches by considering at maximum one missed cleavage site, pyro-Glu formation at the N-terminal Gln, oxidation of methionine, acetylation of the N terminus, and modification of cysteines by acrylamide.

The molecular masses and isoelectric points were calculated by employing the software Compute pI/Mw (available on the World Wide Web).

The protein sequences were analyzed by searching the Pfam HMM data base (available on the World Wide Web) to identify sequence motifs.

Cloning-- The cDNAs were cloned by a combination of reverse transcription and polymerase chain reaction techniques. Reverse transcription was performed on poly(A)+ RNA from the human Jurkat T cell line using oligo(dT) primer. The reverse transcription products were then used as templates for PCR using specific primers according to the di-/trinucleotide sticky end cloning method (Roche Molecular Biochemicals). Primer sequences were as follows: hnRNP A2/B1, 5'-primer TCGAGAGAGAAAAGGAACAGTTC, 3'-primer CTGGTATCGGCTCCTCCCACCATAA; hnRNP R, 5'-primer TCGCTAATCAGGTGAATGGTAATG, 3'-primer CTGCTTCCACTGTTGCCCATAAGTA; p54nrb, 5'-primer TCGAGAGTAATAAAACTTTTAAC, 3'-primer CTGGTATCGGCGACGTTTGTTTGGG; splicing factor ASF-2, 5'-primer TCGGAGGTGGTGTGATTCGTGGC, 3'-primer CTGACACTTTAGCCCATTCTGAAC; splicing factor SRp30c, 5'-primer TCGCGGGCTGGGCGGACGAGCGC, 3'-primer CTGGTAGGGCCTGAAAGGAGAGAAG; transcription factor BTF, 5'-primer TCGGACGGACAGGCGCACCCGCT, 3'-primer CTGTCAGTTTGCCTCATTCTTGGAAGC. The PCR products were treated according to the di-/trinucleotide sticky end cloning method as described by the manufacturer (Roche Molecular Biochemicals) and then introduced into the pET28c vector (Calbiochem-Novabiochem). The cloned cDNAs were sequenced using T3 and T7 sequencing primers.

In Vitro Translation and Cleavage Assay-- The cDNAs were translated in vitro using 35S-labeled methionine with the TNT® coupled reticulocyte lysate system according to the manufacturer's instructions (Promega, Mannheim, Germany). 1 µl of the translation product was cleaved with 3 µl of active lysate or 20 units of caspase-3 (BIOMOL, Hamburg, Germany) in 20 µl of cleavage buffer (25 mM Hepes, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, and the protease inhibitors pefabloc, pepstatin, leupeptin, and aprotinin) for 1 h at 37 °C. For inhibition experiments, 1 µl of 5 mM Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) was added. The cleavage mixture was supplemented with 5 µl of loading buffer (1 µl of glycerol, 1 µl of 10% SDS, 0.25 µl of 2-mercaptoethanol, 0.075 mg of Tris-base, and 0.125 mg of bromphenol blue) and applied to a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was washed, dried, and covered with a BioMaxTM MR film (Eastman Kodak Co.) overnight and then developed.

Active lysate was generated from Jurkat T cells after a 6-h induction of apoptosis with 250 ng/ml alpha CD95 (clone CH11) (Immunotech, Marseille, France) and 1 µg/ml cycloheximide. Subsequently, the cells were washed with phosphate-buffered saline and incubated for 20 min on ice with lysis buffer (25 mM Hepes, 0.1% Chaps, 1 mM dithiothreitol, and the protease inhibitors pefabloc, pepstatin, leupeptin, and aprotinin). Afterward, the cells were homogenized and centrifuged for 5 min at 13,000 units/min (Biofuge fresco; Heraeus Instruments). The supernatant was aliquoted and stored at -70 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Apoptosis-modified Protein Spots-- Apoptosis was induced in Jurkat T cells by treatment with an anti-Fas antibody for 6 h. Since apoptosis is induced in the absence of protein synthesis, cycloheximide was added to the cultures in order to block protein synthesis. Prevention of protein synthesis was a prerequisite to circumvent the induction of stress proteins that are not involved in the primary apoptotic process (17). Two-dimensional gel electrophoresis gels were produced after lysis of the cells and separation of the proteins. A representative two-dimensional gel electrophoresis gel of anti-Fas-treated Jurkat T cells is shown in Fig. 1. Approximately 2000 spots were resolved and detected by silver staining. 10 two-dimensional gel electrophoresis gels of apoptotic cells were compared with 10 two-dimensional gel electrophoresis gels of control cells (Fig. 2). 24 spots were detected in patterns of apoptotic Jurkat T cells that were not present in patterns on nonapoptotic cells. 21 additional spots were observed in the pattern of control cells. Coomassie-stained two-dimensional gel electrophoresis gels were produced for the identification by mass spectrometry because the identification of proteins is not possible by the highly sensitive silver staining technique used. Only 27 of the 45 spots identified in silver-stained gels were detected in Coomassie-stained gels by comparing untreated and Fas-treated Jurkat T cells. Therefore, cytosol, mitochondria, nuclei, and membranes were purified from treated and untreated Jurkat T cells in order to enrich for the proteins that were not detected by Coomassie staining.


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Fig. 1.   Two-dimensional gel electrophoresis gel of Fas-induced Jurkat T cells. Protein spots that were identified in patterns of apoptotic Jurkat T cells by mass spectrometry are indicated. The proteins were detected by silver staining.


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Fig. 2.   Two-dimensional gel electrophoresis gel of Jurkat T cells. Protein spots that were identified in patterns of untreated Jurkat T cells by mass spectrometry are indicated. The proteins were detected by silver staining.

Identified Proteins-- 21 proteins (Table I) were identified in 37 spots by peptide mass fingerprinting after in-gel digestion with trypsin, elution of the generated peptides, and analysis by MALDI-MS. Of the identified proteins, 60 S ribosomal protein P0, hnRNP A2/B1, hnRNP C1/C2, KH-type splicing regulatory protein, p54nrb, and Rho GDI 2 were found at different spot positions in treated and control cells, whereas the other proteins were identified either in treated or in control cells.

                              
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Table I
Identified apoptosis-modified proteins in Jurkat T cells
Indicated are the NCBI accession numbers; protein spots of apoptotic cells (+) and control cells (-), the Mr found in the two-dimensional gel electrophoresis gel, and the theoretical (theor.) mass as well as the observed pI and the theoretical pI. Also specified are the numbers of RNA-binding motifs.

The molecular mass of protein spots in two-dimensional gel electrophoresis gels can usually be determined with an accuracy of about 10%. As expected, the proteins identified in gels of control cells displayed the theoretical mass of the corresponding protein with the exception of hnRNP K. In contrast, five of the 12 proteins identified in gels of apoptotic cells, hnRNP A1, hnRNP A2/B1, hnRNP R, NS1-associated protein 1, and nucleolin, showed a significantly decreased mass of more than 10% compared with the theoretical values. The masses of the remaining seven proteins, 60 S ribosomal protein P0, alpha  NAC, hnRNP C1/C2, KH-type splicing regulatory protein, p54nrb, Rho GDI 2, and SRp30c, corresponded approximately with the theoretical masses. Fortunately, five of these proteins, 60 S ribosomal protein P0, hnRNP C1/C2, KH-type splicing regulatory protein, p54nrb, and Rho GDI 2 were found in patterns of control cells and thus allowed the direct comparison of the masses under both conditions. Using this readout, these proteins were found to exhibit lower masses in the apoptotic pattern, suggesting a modification of these proteins.

We then screened the identified proteins for functional motifs. Surprisingly, 12 proteins contained the RNP motif, and three contained the KH motif (Table I), which are involved in RNA binding and processing (37).

Cleavage Assay-- In order to either verify or determine the cleavage by caspases, the cDNAs of hnRNP A2/B1, hnRNP R, p54nrb, the splicing factor SRp30c, the splicing factor ASF-2, and the transcription factor BTF3 were cloned and expressed in vitro. The proteins were treated with either a lysate of apoptotic Jurkat T cells (not shown), which contained a mixture of active caspases, or with the recombinant purified caspase-3 in the presence or absence of the broad range caspase inhibitor zVAD-fmk (Fig. 3). In most cases, the same cleavage pattern was observed for the proteins treated with the active lysate and caspase-3; however, the cleavage by caspase-3 was more efficient.


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Fig. 3.   Caspase-3 cleavage assay. Proteins were expressed in vitro and labeled using [35S]methionine. Cleavage was carried out by treatment of translated proteins with caspase-3. The assay was performed with and without caspase-3. zVAD-fmk was used to specifically inhibit caspase-3 activity. Note that two translational products were present in the untreated sample of SF ASF-2 and TF BTF3. The smaller fragments (marked with a star) in the caspase-3-treated sample originated from the fragment marked with a star of the untreated sample. The molecular masses are indicated in kDa.

Several bands were observed upon cleavage of p54nrb with caspase-3 (Fig. 3). This was unexpected, since we found only the 52-kDa fragment in apoptotic cells. Inhibition of caspase-3 with zVAD-fmk prevented the generation of all p54nrb fragments, indicating that these fragments were generated by caspase 3. Cleavage of the splicing factor ASF-2 and the transcription factor BTF3 generated two fragments, which probably corresponded to cleavage at one site (Fig. 3). Since the latter factors were identified in control patterns only, cleavage by caspase-3 in vitro suggested that they were also processed in apoptotic cells. As expected from the two-dimensional gel electrophoresis results, cleavage of the splicing factor SRp30c by caspase-3 was not observed (Fig. 3). This indicated either a modification of SRp30c other than cleavage or that cleavage occurred very close to the N terminus of the protein, because the C-terminal end was identified by the mass spectrometrical analysis. The 49-kDa fragment detected in caspase-3 cleavage assays with hnRNP R (Fig. 3) confirmed the two-dimensional gel electrophoresis data. Only hnRNP A2/B1, which was fragmented in apoptotic cells, could neither be cleaved by the active lysate (not shown) nor by caspase-3 (Fig. 3). Therefore, cleavage of hnRNP A2/B1 by caspase-3 can be excluded due to the high efficiency of the enzyme. hnRNP A2/B1 might be cleaved by another protease in vivo, which is not active under the conditions used in the in vitro cleavage assay.

Prediction of Cleavage Sites-- The sites at which caspases cleaved the substrates can be calculated by taking into consideration that these enzymes cleave target proteins specifically after aspartic acids. Further parameters necessary for the prediction of cleavage sites were the sequence coverage of the peptide mass fingerprints, the difference of the theoretical and the detected molecular masses, and the pI values of the proteins that were identified in patterns of apoptotic cells. For the splicing factor ASF-2 and the transcription factor BTF, which were not found in patterns of apoptotic cells, the cleavage site could be predicted by means of the in vitro cleavage assay (Fig. 4, Table II).


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Fig. 4.   Schematic representation of the putative cleavage sites. The putative cleavage sites are shown by arrows. The sequence coverage of the peptide mass fingerprints analyzed from spots of apoptotic cells are displayed by dotted lines. RNP motifs are illustrated as gray boxes, KH domains as black boxes, and the nuclear targeting sequence of hnRNP A1 as M9.

                              
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Table II
Prediction of cleavage sites
The putative cleavage sites for caspases were calculated by the theoretical masses of fragments generated by cleavage after aspartic acids. The difference between the theoretical mass and the observed mass was <10% and 0.5 for the pI. In addition, the sequences covered by the peptide mass fingerprinting were considered. P indicates the values of fragments derived from patterns of apoptotic cells, N from control patterns. C indicates that the found mass was determined by the cleavage assay with a mass accuracy of ±1 kDa. Splicing factor and transcription factor are abbreviated by SF and TF, respectively.

Using this method, one cleavage site could be calculated for Rho GDI 2 (Table II), which has previously been shown to be cleaved by caspase-3 (38). This example suggested that the applied procedure is reliable and useful for calculating caspase cleavage sites. The caspase-generated fragment of nucleolin could be evaluated by cleavage at the sequence TEID and a second cleavage at the sequence AMED or GEID. From the factors that were not yet known to be cleaved by caspases, one cleavage site very close to the C terminus was calculated for p54nrb (Table II). Cleavage or any other modification could be assumed for the splicing factor SRp30c and the 60 S ribosomal protein P0 due to the identification in two-dimensional gel electrophoresis gels of apoptotic cells. If the splicing factor SRp30c was cleaved by caspases, the only possible site must be near the N terminus, because the peptide mass fingerprint included the peptide of the C-terminal end (Table II). Three putative cleavage sites near the N terminus or C terminus can be calculated for the 60 S ribosomal protein P0. However, the cleavage of the splicing factor SRp30c and 60 S ribosomal protein P0 could not be proven unequivocally by the observed molecular mass, pI, and cleavage assay. Two alternative putative sites were found for the cleavage of hnRNP A1, three alternative sites near the N terminus for hnRNP A2/B1, three alternative sites near the C terminus for NS1-associated protein 1, and seven alternative sites near the N terminus for KH-type splicing regulatory protein (Table II). hnRNP C1/C2 was cleaved either at a site near the N terminus or three potential sites near the C terminus. Two cleavages can be predicted for hnRNP R with two potential sites in each case.

According to the fragment patterns obtained after cleavage by caspase-3 in vitro, transcription factor BTF3 and the splicing factor ASF-2 were cleaved only once (Fig. 3, Table II). The possible cleavage site for BTF3 could be predicted, whereas four neighboring putative cleavage sites in ASF-2 did not allow a precise cleavage site prediction (Table II).

The RNP consensus sequence of the RNP motif is composed of two short sequences, RNP1 and RNP2, and a number of other conserved amino acids with a total length of about 70 amino acids (37). We therefore determined whether the cleavage by caspases would affect the RNP motifs. According to the predicted cleavage sites, the RNP motifs of hnRNP A1, hnRNP C1/C2, hnRNP R, NS1-associated protein 1, p54nrb, and SRp30c would not be affected by cleavage, whereas one of the two RNP motifs of hnRNP A2/B1 and splicing factor ASF-2 and two of the four RNP motifs of nucleolin could be cleaved (Fig. 4).

Cleavage within the KH motifs of the KH-type splicing regulatory protein was not predicted.

Another interesting prediction concerns the nuclear localization sequence M9 (39). This part of the hnRNP A1 would be separated from the protein, implying functional consequences of the caspase cleavage.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we describe the identification of proteins that are modified in the course of CD95 (Fas/Apo-1)-induced apoptosis. Among these proteins, alpha  NAC, hnRNP A0, hnRNP A2/B1, hnRNP A3, hnRNP K, hnRNP R, KH-type splicing regulatory protein, NS1-associated protein 1, p54nrb, poly(A)-binding protein 4, and splicing factors ASF-2 and SRp30c were not known to be modified during apoptosis. However, we also identified proteins that were previously found to be modified during apoptosis by a proteome approach using the Burkitt lymphoma B cell line HL-60 and IgM as an inducer of apoptosis (16, 40, 41). These proteins included hnRNP A1, hnRNP C1/C2, FUSE-binding protein, nucleolin, Rho GDI 2, and the transcription factor BTF3. The fact that we identified proteins that were not found to be modified in HL-60 cells might resemble differences in either the cell type or the inducers of apoptosis used. Interestingly, the spot position of hnRNP A1, nucleolin, and Rho GDI 2 differed between apoptotic Jurkat T cells and apoptotic HL-60, suggesting that these proteins were differently modified during apoptosis. Thus, proteome approaches are useful to identify apoptosis-modified proteins and offer the possibility to define differences regarding cell types and signaling pathways.

Using in vitro cleavage assays, we demonstrated that of the newly identified factors, p54nrb, splicing factor ASF-2, transcription factor BTF3, and hnRNP R are substrates for caspase-3. While hnRNP R, transcription factor BTF3, and splicing factor ASF-2 were cleaved at one site, cleavage of p54nrb by caspase-3 produced a highly complex pattern of at least seven fragments. From these seven fragments, only one was found in patterns of apoptotic cells, suggesting that the other fragments either were not detected in two-dimensional gel electrophoresis gels or were not generated in apoptotic cells in vivo. No cleavage by caspase-3 was observed using hnRNP A2/B1 as the substrate, although this protein was identified in patterns of apoptotic cells with a clearly reduced mass compared with the mass of the control spot. It is thus possible that either a caspase with a different cleavage specificity than caspase-3 or any other protease activated during apoptosis cleaved hnRNP A2/B1 in vivo. If the latter explanation is correct, hnRNP A2/B1 would add another example to a growing list of substrates that are cleaved during apoptosis by proteases other than caspases (42). Another substrate for which we could not confirm cleavage by caspases was splicing factor SRp30c. This protein was identified in patterns of apoptotic cells but not in control patterns; thus, a direct comparison of the size was not possible. However, if the modification involved cleavage, this probably occurred very close to the N terminus, since we identified peptides from amino acid 9 to the end of the protein by mass spectrometry. A possible cleavage site thus could involve the sequence GWAD at position 5, which would meet the requirement of a caspase cleavage site (43).

The most striking common feature of the apoptosis-modified proteins identified was the presence of an RNA-binding motif in 15 out 21 proteins. 12 of these 15 proteins, with the exception of FUSE-binding protein, nucleolin, and poly(A)-binding protein 4, have likely functions in RNA splicing. The proteins identified in this study fall into two major functional groups: 1) splicing factors like SRp30c, ASF-2, p54nrb, and KH-type splicing regulatory protein and 2) hnRNPs, involved in RNA modification or stabilization. We could not recognize a common rule regarding caspase cleavage in or outside the RNP motifs. Caspase cleavage definitely occurs in the RNP motif of hnRNP A2, nucleolin, and splicing factor ASF-2 (Fig. 4), suggesting that in these factors cleavage could affect binding of RNA. In all of the other factors, the RNP motif would not be affected by caspase cleavage. An interesting finding was that in hnRNP A1 the M9 motif possibly would be separated by caspase cleavage from the rest of the protein. Since this motif is important for the import of proteins into the nucleus (44), cleavage could affect protein targeting in a direct way. In a search for the abundance of the RNP and KH motif in human proteins, 213 and 28 proteins, respectively, out of 15,683 (1.5%) were identified (at the European Bioinformatics Institute site on the World Wide Web). Thus, the preferred identification of RNA-binding motifs containing proteins is clearly significant. The question arises, what is the significance of such a tremendous modification of RNA-modifying proteins during apoptosis? The current concept of apoptosis defines the activation of caspases as the "point of no return," the point from which the cell is definitely committed to undergo apoptosis. If a cell is going to die anyway, why should this cell modify RNA? First, it might be important to degrade large protein-RNA complexes in order to allow an "ordered" destruction and clearance of the cell. And second, situations might exist, where caspases are activated, but cells do not die due to overexpression of inhibitors of apoptosis. The latter situation has been found in certain cells that require mitochondrial function also when apoptosis is induced by death receptors (45). Thus, it is possible that regulation of splicing under these circumstances influences the apoptotic process in the long term (e.g. by producing inhibitors or activators of apoptosis).

One example of an alternatively spliced factor with pro- or antiapoptotic activities is Bcl-X (46). Bcl-XL exerts antiapoptotic activities, whereas Bcl-XS expressed from an alternatively spliced form of the Bcl-X gene induces apoptosis (47). Although Bcl-XL is expressed in most cells in vivo, apoptosis induction (e.g. during thymic selection) is accompanied by the expression of Bcl-XS (48). One possible explanation is that alternative splicing not only occurs dependent on the cell type, but it is also induced by proapoptotic triggers.

Yet another example is given by a possible regulation of alternative splicing of caspase-2. The long form of caspase-2, also known as Ich-1L, promotes apoptosis, while Ich-1S inhibits apoptosis (49). Strikingly, splicing of caspase-2 RNA is controlled by factors that were found to be modified during apoptosis in this study. hnRNP A1 and A2/B1 function as splicing silencers (50, 51), which antagonize the alternative splicing activity of the splicing factors SF2/ASF and SC35 (52-55). The same is true for the splicing factor ASF-2, which is an alternative splice variant of SF2/ASF (56). These proteins differ in their C-terminal domains. ASF-2 lacks the RS domain, which is essential for some but not all functions of SR proteins. ASF-2 acts as a splicing repressor and may compete with the splicing enhancer SF2/ASF. The relative amount of Ich-1L, which promotes apoptosis, is increased by SF2/ASF and SC35, whereas hnRNP A1 increases the relative amount of Ich-1S, which prevents apoptosis (57). Thus, cleavage of either of these factors could result in production of alternatively spliced forms of caspase-2, which either promote or prevent apoptosis.

What could be the advantage of regulating apoptosis by RNA modification? In particular, the regulation of apoptosis could require a fast response, which is given by modifying RNA rather than by de novo synthesis of mRNA. Specific mRNAs can be stored as mRNA-protein complexes, and in response to a stimulus the masking proteins are removed or modified, and the mRNA is translated (58). Other mRNAs could be generated by apoptosis-specific alternative splicing, resulting either in prevention or promotion of apoptosis.

More than 60 substrates for caspases, which are either activated, inactivated, destroyed, or transported to a different cellular compartment due to cleavage have already been described (9). In this study, we have identified several caspase substrates involved in modulating RNA stability or processing. Although the role of RNA modification in the regulation of apoptosis is not well understood, the simple fact that we identified mainly RNA-modifying proteins in a proteomics approach to identify new regulators of apoptosis suggests that these proteins are important. It will certainly be a challenge to unravel such a complex process as alternative splicing in the context of apoptosis, where major factors are processed and possibly modified in their function.

    FOOTNOTES

* This work was supported by the grant 12260 from the Bundesministerium für Bildung und Forschung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 49-30-450-78024; Fax: 49-30-450-78925; E-mail: rudel@mpiib-berlin.mpg.de.

Published, JBC Papers in Press, May 14, 2001, DOI 10.1074/jbc.M101062200

    ABBREVIATIONS

The abbreviations used are: MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; BSA, bovine serum albumin; hnRNP, heterogeneous nuclear ribonucleoprotein; zVAD-fmk, Z-Val-Ala-DL-Asp-fluoromethylketone; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GDI, guanine nucleotide dissociation inhibitor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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