 |
INTRODUCTION |
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 |
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
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
-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
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 |
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.

View larger version (91K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (110K):
[in this window]
[in a new window]
|
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.
View this table:
[in this window]
[in a new window]
|
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,
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.

View larger version (38K):
[in this window]
[in a new window]
|
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).

View larger version (22K):
[in this window]
[in a new window]
|
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.
|
|
View this table:
[in this window]
[in a new window]
|
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 |
Here we describe the identification of proteins that are modified
in the course of CD95 (Fas/Apo-1)-induced apoptosis. Among these
proteins,
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.