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La Autoantigen Is Cleaved in the COOH Terminus and Loses the
Nuclear Localization Signal during Apoptosis*
Received for publication, May 1, 2000, and in revised form, July 25, 2000
Published, JBC Papers in Press, July 26, 2000, DOI 10.1074/jbc.M003673200
Koichi Ayukawa‡, Shun’ichiro Taniguchi‡, Junya Masumoto‡, Shigenari Hashimoto‡,
Haritha Sarvotham‡, Astushi Hara§, Toshifumi Aoyama§, and Junji Sagara‡
¶
From the Departments of ‡Molecular Oncology and Angiology and §Aging Biochemistry, Research Center on Aging and
Adaptation, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, 390-8621 Nagano, Japan
La autoantigen is a 47-kDa nuclear protein that binds
to nascent polymerase III transcripts and a number of
viral RNAs. We show that La protein was cleaved to
generate a 43-kDa fragment during apoptosis of human
leukemic HL-60 cells treated with camptothecin or eto-
poside. Immunofluorescence microscopy showed that
the La protein level was increased in the cytoplasm
during apoptosis of HL-60 cells. In addition, UV irradi-
ation of HeLa cells led to the cleavage and redistribution
of La protein upon apoptosis. Several lines of evidence
show that La protein is cleaved by caspase-3 or closely
related proteases at Asp-374 in the COOH terminus.
When the full-length (La) and COOH-terminally trun-
cated (La⌬C374) forms of La protein were expressed as
fusion proteins with green fluorescence protein (GFP),
GFP-La⌬C374 was predominantly cytoplasmic, whereas
GFP-La was localized in the nucleus. These results sug-
gest that La protein loses the nuclear localization signal
residing in the COOH terminus upon cleavage and is
thus redistributed to the cytoplasm during apoptosis.
Apoptosis is accompanied by disorganization of the nuclear
architecture, cytoskeleton, and cell membrane. Proteolytic
cleavage of key substrates is an important biochemical mech-
anism underlying the apoptotic process, and interleukin-1

-
converting enzyme-like proteases have been reported to play
crucial roles as mediators of apoptosis (1–3). Identifying sub-
strates of interleukin-1

-converting enzyme or interleukin-1

-
converting enzyme-like proteases and determining cleavage
sites are important to understand the apoptotic process.
A variety of antitumor drugs have been shown to induce
apoptosis and to cause changes in nuclear morphology in rap-
idly proliferating cells, lymphoid tissues, and tumors (4, 5).
Camptothecin and etoposide, which are antitumor drugs, in-
duce apoptosis of HL-60 cells in addition to numerous tumors
(6–8). We developed several monoclonal antibodies (mAbs)
1
to
examine changes in nuclear architecture during this process.
We screened for changes in the expression and/or localization of
components of the cytoskeleton and nuclear matrix in associa-
tion with apoptosis of HL-60 cells.
Here, we report that the nuclear protein La is cleaved by
caspase-3-like protease in the COOH terminus and that its
level is increased in the cytoplasm during apoptosis. In cells, La
protein binds to the oligo(U) 3⬘ termini of nascent polymerase
III transcripts and viral RNAs (9–17). La protein is well known
as an autoantigen, and sera from patients with rheumatoid
diseases such as systemic lupus erythematosus frequently con-
tain antibodies directed against La protein (18, 19). La antigen
was reported to exhibit changes in its distribution in apoptotic
cells (20, 21). Apoptotic cell antigens have been identified as
the targets of autoantibodies in autoimmune diseases, and it
has been proposed that apoptotic cells are a primary source of
autoantigens (20, 22). Our findings provide insight into the
mechanism of redistribution of La protein during apoptosis of
various types of cells and the production of autoantibodies
against La protein.
EXPERIMENTAL PROCEDURES
Cell Culture Conditions, Reagents, and Irradiation—Mouse myeloma
P3-X63-Ag8.653 cells that were used for production of hybridoma were
obtained from the RIKEN Gene Bank. HL-60, Jurkat, U937, and HeLa
cells were obtained from American Type Culture Collection. Leukemia
cells were grown in RPMI 1640 medium supplemented with 10% fetal
bovine serum in an atmosphere of 95% air and 5% CO
2
. Camptothecin
(TopoGEN) and etoposide (Nippon-Kayaku) were dissolved in a 1:1 (v/v)
mixture of dimethyl sulfoxide and ethanol and stored at ⫺20 °C until
used. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 5% fetal bovine serum. Subconfluent monolayer
cultures of HeLa cells were washed twice with PBS (2.7 mM KCl, 1.5 mM
KH
2
PO
4
, 137 mM NaCl, and 8 mM Na
2
HPO
4
) and irradiated in PBS
with 100 J of 254 nm UV light/m
2
(Spectrolinker XL-1500, Spectronics
Corp., Westbury, NY). The anti-poly(ADP-ribose) polymerase (PARP)
C-2-10 antibody was obtained from Oncogene Research Products
(Cambridge).
Preparation of a Triton X-100-insoluble Fraction of HL-60 Cells and
Production of mAb—After washing with PBS, HL-60 cells were ex-
tracted on ice for 5 min with 0.5% Triton X-100 in cytoskeleton buffer
(10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl
2
,1
mM EGTA, 4 mM vanadyl riboside complex, and 1 mM phenylmethyl-
sulfonyl fluoride). The insoluble fraction was collected by centrifugation
at 600 ⫻ g for 5 min. For immunization, chromatin was removed from
the Triton X-100-insoluble material by DNase I (Sigma) digestion,
followed by 0.25 M ammonium sulfate extraction as described by Fey
and Penman (23). The chromatin-depleted insoluble materials were
mixed with Freund’s complete adjuvant for the first immunization or
with Freund’s incomplete adjuvant for booster immunizations. Spleen
cells were collected from immunized BALB/c mice and were fused with
mouse myeloma cells by the polyethylene glycol method (24).
Identification of Antigens by Immunoscreening of the cDNA Li-
brary—Total RNA was isolated from HL-60 cells, and mRNA was
purified on an oligo(dT)-cellulose column (25). cDNAs were synthesized
using random hexadeoxynucleotides as the primer and cloned into the
EcoRI site of
gt11 arms according to the manufacturer’s instructions
(Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
¶
To whom correspondence should be addressed. Tel.: 81-263-37-
2723; Fax: 81-263-37-2724; E-mail: sagara@sch.md.shinshu-u.ac.jp.
1
The abbreviations used are: mAbs, monoclonal antibodies; PBS,
phosphate-buffered saline; PARP, poly(ADP-ribose) polymerase; PIPES,
1,4-piperazinediethanesulfonic acid; TUNEL, terminal deoxynucleoti-
dyltransferase-mediated dUTP nick end labeling; GFP, green fluores-
cent protein; DE-MALDI-TOF-MS, delayed extraction matrix-assisted
laser desorption ionization time-of-flight mass spectrometry; NLS, nu-
clear localization signal.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 44, Issue of November 3, pp. 34465–34470, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 34465
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The
gt11 cDNA libraries were immunoscreened with this mAb (24) as
described (25). The cloned cDNAs were amplified by polymerase chain
reaction using
gt11 forward and reverse primers and introduced into
the vector pGEX4T-1 (Amersham Pharmacia Biotech) for expression as
a glutathione S-transferase fusion protein. The glutathione S-transfer-
ase fusion proteins were subjected to Western blot analysis with the
mAb to exclude false-positive cDNA clones. The polymerase chain re-
action product of the cDNA clone was directly sequenced by the dideoxy
chain termination method (26). To determine the region of La protein
recognized by mAb 24 more precisely (see “Results”), the coding se-
quence was amplified by polymerase chain reaction, inserted into
pGEX4T-1, and expressed as a glutathione S-transferase fusion
protein.
Immunofluorescence Microscopy—HL-60 cells were collected by cen-
trifugation at 600 ⫻ g for 5 min, placed on 14-well Teflon-coated slide
glasses (Cel-Line/Erie Scientific, Portsmouth, NH), and fixed with 70%
ethanol or acetone/methanol (1:1) for 15 min. After soaking in PBS
containing 1% bovine serum albumin for 15 min, samples were incu-
bated with the first antibody at room temperature for 60 min and then
stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG
(Dako Japan, Kyoto, Japan) in 20
g/ml propidium iodide. Samples
were examined using a fluorescence microscope (Axioskop, Carl Zeiss,
Oberkochen, Germany).
Apoptotic cells were determined by the terminal deoxynucleotidyl-
transferase-mediated dUTP nick end labeling (TUNEL) method (27).
DNA strand breaks were demonstrated by labeling 3⬘-OH termini with
fluorescein isothiocyanate-labeled deoxyuridine using an in situ apo-
ptosis detection kit (Takara, Kyoto). After TUNEL staining, cells were
immunostained with anti-La mAb. In this case, tetramethylrhodamine
isothiocyanate-conjugated goat anti-mouse IgG (Dako Japan) was used
as the second antibody.
In Vitro Cleavage with Recombinant Caspase-3—HL-60 cells (1 ⫻
10
6
) were washed with Krebs-Ringer buffer and lysed on ice with 1 ml
of ice-cold lysis buffer containing 10 m
M HEPES/KOH (pH 7.4), 2 mM
EDTA, 5 mM dithiothreitol, 1% Nonidet P-40, and the protease inhibi-
tors phenylmethylsulfonyl fluoride, antipain, and leupeptin (28). The
protein concentration of the cell lysate was adjusted to 1 mg/ml. Ali-
quots of 18
l of cell lysate were incubated at 37 °C in the presence or
absence of 2
l of 0.37 mg/ml (3300 units/ml) recombinant caspase-3
(Medical and Biological Laboratories Co., LTD, Nagoya, Japan) accord-
ing to the manufacturer’s instructions.
Production of Antibodies against the NH
2
and COOH Termini of
La—An NH
2
-terminal peptide (
1
MAENGDNEKMAALEA
15
) and a
COOH-terminal peptide (
388
ETDKEEPASKQQKTE
402
) of La were syn
-
thesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide
synthesis (Takara). A cysteine residue was added to the NH
2
terminus
of each of the synthetic peptides for coupling to bovine serum albumin.
The peptides were coupled to bovine serum albumin using maleimido-
benzoic acid hydroxysuccinimide ester as a linker (29). The peptide-
coupled bovine serum albumin was used to immunize New Zealand
White rabbits, and antisera were obtained.
Expression of the Full-length and COOH-terminally Truncated
Forms of La Proteins as Fusion Proteins with GFP—cDNAs of full-
length (La) and COOH-terminally truncated (La⌬C374) forms of La
proteins were synthesized by reverse transcription and polymerase
chain reaction. An antisense primer of La mRNA (5⬘-TAAACTACTG-
GTCTCCAGCA-3⬘) was used for reverse transcription. cDNAs of La and
La⌬C374 were amplified by polymerase chain reaction using the sense
primer 5⬘-CGGAATTCATGGCTGAAAATGGTGATAAT-3⬘ and the an-
tisense primers 5⬘-ACGCGTCGACCTACTGGTCTCCAGCACCAT-3⬘
and 5⬘-ACGCGTCGACGCTAATCATGTTCATCATGT-3⬘, respectively.
Each cDNA was digested with EcoRI and SalI and cloned into the
pEGFP-c2 mammalian expression vector (CLONTECH). Exponentially
growing COS-7 cells were plated in 6-well tissue culture dishes for
Western blot analysis or on coverslips for immunofluorescence staining
and incubated for 24 h at 37 °C. Each construct was transfected into
COS-7 cells by lipofection using Transome
TM
(Wako, Osaka, Japan)
according to the manufacturer’s instructions.
Determination of Molecular Mass by Delayed Extraction Matrix-as-
sisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
(DE-MALDI-TOF-MS)—Hybridoma was cultured in protein-free hybri-
doma medium Protein-free hybridoma medium-II (Life Technologies,
Inc.), and anti-La mAb was purified from culture fluid using protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech). The purified an-
ti-La mAb was conjugated to CNBr-activated Sepharose 4B (Amersham
Pharmacia Biotech). HL-60 cells that were treated with 10
g/ml eto-
poside for 12 h were lysed in 150 mM NaCl, 50 mM Tris-HCl (pH 8.0),
and the protease inhibitors phenylmethylsulfonyl fluoride, aprotinin,
antipain, pepstatin A, and leupeptin. The cell lysate was clarified by
centrifugation at 10,000 ⫻ g for 20 min and incubated with anti-La
mAb-conjugated Sepharose 4B for1hat4°C.After elution with 0.1
M
glycine (pH 2.5), the affinity-purified proteins were concentrated and
desalted using a centrifugal filter device (Microcon YM-30, Millipore
Corp.). DE-MALDI-TOF-MS was carried out on a Voyager Elite XL
Biochemical Workstation (6.5-m flight length linear mode; Perseptive
Biosystems, Framingham, MA). Sinapinic acid was used as a matrix. A
nitrogen laser (337 mm) was used for ionization. Acceleration voltage
was set 20,000 V; grid voltage was set at 74% of the acceleration
voltage; and guide wire voltage was 0.05% of the acceleration voltage.
Delay time was 250 ns.
RESULTS
Production of Anti-La mAb—To examine changes in nuclear
architecture during apoptosis, we developed mAbs against nu-
clear matrix proteins (23) in apoptotic HL-60 cells. Labeling
with one mAb, designated mAb 24, was examined using immu-
nofluorescence microscopy, and marked changes in HL-60 cells
during apoptosis were observed. We immunoscreened
gt11
HL-60 cDNA libraries with mAb 24 and isolated one cDNA
clone encoding a polypeptide that was identical to La protein
from amino acids 320 to 393. La is a nuclear protein composed
of 408 amino acids with a predicted molecular mass of 46,837
Da (30, 31). mAb 24 recognized a polypeptide with a molecular
mass of ⬃47 kDa upon Western blotting (Fig. 1) and stained
the nuclei of various human cells upon immunofluorescence
microscopy.
Cleavage of La Protein upon Apoptosis—Western blot anal-
ysis of cell lysates with anti-La mAb revealed extra polypep-
tides in addition to the native 47-kDa polypeptide of La in
HL-60 cells treated with camptothecin (Fig. 1a) or etoposide
(data not shown). A 43-kDa fragment was prominent in lysates.
PARP is a well documented caspase-3 substrate and is cleaved
in the early stages of apoptosis (32–34). We compared La pro-
tein with PARP in HL-60 cells and found that after incubation
with camptothecin for 12 h, ⬎80% of PARP was cleaved (data
not shown), but only ⬃35–40% of La protein was cleaved (Fig.
1a). This result indicates that La protein is not as good a
substrate for caspases as PARP. Similarly, camptothecin in-
duced apoptosis of human monocytic leukemic U937 cells and
T-cell lymphoma Jurkat cells, where the 43-kDa fragment was
generated (data not shown).
Cellular Localization of La Protein in Non-apoptotic and
Apoptotic HL-60 Cells—Immunofluorescence analysis with an-
ti-La mAb revealed marked changes in HL-60 cells treated
with camptothecin or etoposide. In non-apoptotic cells, La was
predominantly nuclear. However, in apoptotic cells, nuclear La
protein levels were decreased, whereas cytoplasmic levels were
increased (Fig. 1b). TUNEL staining clearly demonstrated that
the redistribution of La protein was associated with DNA frag-
mentation (Fig. 1c). Similar findings were observed in Jurkat
and U937 cells, where La protein levels were increased in the
cytoplasm during apoptosis induced by the antitumor drugs
(data not shown).
TUNEL staining and various morphological features such as
reduction in cell volume and cytoplasmic blebbing were used to
identify apoptotic cells. After treatment with camptothecin or
etoposide, apoptotic cells were found to increase as shown in
Fig. 2. However, in some cases where cell fragmentation had
occurred, immunofluorescence staining was not seen with
TUNEL, and 50% of these cells were permeable to trypan blue.
Furthermore, La staining was seen in TUNEL-negative cells
that appeared to be highly fragmented, and the levels of La
staining were much lower than those in TUNEL-positive cells.
These results indicate that some populations of apoptotic cells
go on to late stages of apoptosis or undergo secondary necrosis
in antitumor drug-treated HL-60 cells.
Cytoplasmic La staining was seen in most of the TUNEL-
Apoptosis-associated Cleavage and Redistribution of La Proteins34466
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positive cells (Fig. 2). Here, we must emphasize that cytoplas-
mic La protein levels were variable among TUNEL-positive
cells, and La staining was still seen in the nuclei of most of
these cells (Fig. 1c). La protein exhibited two changes upon
apoptosis, i.e. proteolytic cleavage and nuclear-to-cytoplasmic
redistribution. We next investigated the relationship between
the generation of the 43-kDa polypeptide and the nuclear-to-
cytoplasmic redistribution of La antigen.
Cleavage and Redistribution of La Protein in Apoptotic HeLa
Cells—To investigate whether these changes in La protein seen
in apoptotic leukemia cells occurred in adherent cells upon
apoptosis, we analyzed the apoptotic process in HeLa cells. UV
irradiation of HeLa cells led to nuclear condensation and mor-
phological changes characteristic of apoptosis after ⬃6–24 h.
La protein was predominantly nuclear in non-apoptotic HeLa
cells, but it translocated to the cytoplasm upon apoptosis (Fig.
3b). The cleavage product of the 43-kDa polypeptide was in-
creased during apoptosis induced by UV irradiation, and a good
correlation was observed between cleavage of La and the num-
ber of cells exhibiting cytoplasmic La staining (Fig. 3c). We
compared La protein with PARP in HeLa cells and found that
La protein was not as good a substrate for caspases as PARP
(Fig. 3a), as found in HL-60 cells. These results in HeLa cells
were consistent with those in leukemia cells, indicating that
the cleavage and redistribution of La protein are common in
various types of cells during apoptosis.
Cleavage of La Protein in the COOH Terminus—The native
47-kDa La polypeptide is cleaved to generate a 43-kDa frag-
ment during apoptosis. This result indicated that La protein is
cleaved at ⬃30–40 amino acids downstream from the NH
2
terminus or upstream from the COOH terminus. We developed
antisera against the NH
2
- and COOH-terminal peptides of La
protein in rabbits (Fig. 4a). As shown in Fig. 4b, the anti-
FIG.1.Cleavage of La protein and cellular localization of La
protein in apoptotic HL-60 cells. a, HL-60 cells were incubated with
1
M camptothecin for the indicated times. The cell lysates were sub-
jected to Western blot analysis with anti-La mAb 24. La protein and the
43-kDa fragment are indicated by the arrow and arrowhead, respec-
tively. Numbers to the left correspond to migration positions of protein
markers (in kilodaltons). b, HL-60 cells were incubated with or without
1
M camptothecin for 6 h. Untreated (row A) or camptothecin-treated
(row B) HL-60 cells were collected and immunostained with anti-La
mAb 24 (green). Nuclei were stained with propidium iodide (red). In
untreated cells, the distribution of La protein was predominantly nu-
clear (row A). In some camptothecin-treated cells, the nucleus was
condensed, and the La protein level was increased in the cytoplasm (row
B). Bar ⫽ 5
m. c, the distribution of La protein in camptothecin-
treated HL-60 cells was determined by immunostaining with anti-La
mAb 24 (red), and DNA fragmentation in situ was detected by the
TUNEL method (green).
FIG.2.Kinetic studies on apoptosis and redistribution of La
protein. HL-60 cells were incubated with camptothecin (a) or etoposide
(b) for the indicated times as described in the legend to Fig. 1. Cells
were collected, and the distribution of La protein was determined by
immunostaining with anti-La mAb 24. Cells exhibiting cytoplasmic La
staining were counted. TUNEL-positive cells were solely those cells
stained by the TUNEL method as described in the legend to Fig. 1, and
apoptotic cells were TUNEL-positive cells and TUNEL-negative cells
with apoptotic morphology (blebbing and cell fragmentation). Cells
exhibiting both cytoplasmic La staining and TUNEL-positive cells were
also counted.
FIG.3.Cleavage and redistribution of La protein in apoptotic
HeLa cells. Monolayer cultures of HeLa cells were irradiated with UV
and incubated in PBS. a, cell lysates were subjected to Western blot
analysis with anti-La mAb 24 as described in the legend to Fig. 1. b,
UV-irradiated cells were fixed and immunostained with anti-La mAb 24
(green). Nuclei were stained with propidium iodide (red). In the apo-
ptotic cells (arrowheads) showing nuclear condensation and membrane
blebs, the La protein level was decreased in the nucleus and increased
in the cytoplasm. Bar ⫽ 20
m. c, shown is the correlation between
cleavage of La and the number of cells exhibiting cytoplasmic La stain-
ing. The results of Western blotting with anti-La mAb 24 were analyzed
using NIH Image. Apoptotic cells were determined by nuclear conden-
sation and morphological changes characteristic of apoptosis.
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COOH-terminal peptide antibody recognized the native 47-kDa
polypeptide, but not the cleaved 43-kDa polypeptide, whereas
the anti-NH
2
-terminal peptide antiserum recognized both the
47- and 43-kDa polypeptides. These results demonstrate that
La protein is cleaved at ⬃30–40 amino acids upstream from
the COOH terminus (Fig. 4a).
The DEXD motif of the caspase-3 cleavage site (35, 36) is
present in the COOH terminus of human La protein,
368
DEH
-
DEHD
374
(Fig. 4a). As mentioned above, mAb 24 used in this
study was identified as an anti-La mAb recognizing a polypep-
tide of La protein from amino acids 320 to 393. The putative
caspase-3 cleavage site(s) was present from amino acids 320 to
393. We then expressed a polypeptide of La from amino acids
320 to 371 as a fusion protein with glutathione S-transferase
using the pGEX4T-1 expression vector. Western blotting
showed that anti-La mAb 24 recognized the fusion protein
(data not shown), indicating that the epitope of anti-La mAb 24
is present from amino acids 320 to 371 of La protein.
We tested whether exogenous caspase-3 would cleave La
protein as seen during apoptosis. Incubation of untreated cell
lysate with recombinant caspase-3 resulted in cleavage of the
47-kDa La polypeptide to a 43-kDa fragment, identical in size
to the fragment observed during apoptosis (Fig. 5a). We next
examined the effects of caspase inhibitors on cleavage of La
protein in intact cells. A specific inhibitor of caspase-3, acetyl-
DEVD-aldehyde, decreased generation of the 43-kDa fragment
in HL-60 cells treated with antitumor drugs (Fig. 5b). In con-
trast, the interleukin-1

-converting enzyme inhibitor acetyl-
YVAD-aldehyde did not inhibit cleavage of La protein.
There are two possible caspase-3 cleavage sites (Asp-371 and
Asp-374) in the COOH terminus of La protein. To map the
cleavage site, we isolated La protein and its fragment from
etoposide-treated HL-60 cells by anti-La mAb affinity chroma-
tography and determined the molecular mass by DE-MALDI-
TOF-MS (Fig. 6). The molecular masses of the native La
polypeptide and the 43-kDa polypeptide are 46,831 ⫾ 31 and
43,182 ⫾ 44 Da (n ⫽ 5), respectively. Based on the amino acid
sequence, the molecular mass of the native La protein is 46,837
Da, and those of two possible fragments are 42,816 Da for
Asp-371 and 43,197 Da for Asp-374. This result indicates that
La protein is cleaved after Asp-374 by caspase-3 or a caspase-
3-like protease in apoptotic cells.
Expression of COOH-terminally Truncated Forms of La Pro-
tein—Simons et al. (37) assigned the NLS to the COOH-termi-
nal polypeptide from amino acids 383 to 401 by microinjection
of in vitro translated La protein into Xenopus laevis oocytes. To
evaluate the effects of the COOH-terminal truncation of La
protein on its localization, we expressed the full-length or trun-
cated form of La protein as a fusion protein with GFP using the
pEGFP-c2 expression vector. Since we identified the site of
cleavage, we constructed plasmids to fuse the full-length (La)
or truncated (La⌬C374) form of La protein to the COOH ter-
minus of GFP. When each construct was transfected into
COS-7 cells, each fusion protein of the expected size was ex-
pressed (Fig. 7a). Immunofluorescence analysis of these cells
showed that the GFP fusion protein with full-length La protein
(GFP-La) was localized in the nucleus. In contrast, GFP-
La⌬C374 was distributed in the cytoplasm (Fig. 7b). Nuclear
green fluorescence staining was seen in COS-7 cells expressing
GFP-La⌬C374, and almost the same levels of green fluores-
cence were seen in those expressing GFP (Fig. 7b). Image
analysis showed that the intensity of nuclear green fluores-
cence was less (⬍5%) than that of cytoplasmic staining in both
cases for GFP and GFP-La⌬C374. The reason for the leakage of
GFP and GFP-La⌬C374 to the nucleus was uncertain.
When COS-7 cells expressing GFP-La were irradiated with
UV, nuclear-to-cytoplasmic redistribution of green fluorescence
was seen in some cells. Furthermore, a cleaved polypeptide
that was almost the same size as GFP-La⌬C374 was detected
by Western blot analysis (data not shown). These observations
FIG.4.Cleavage of La protein in the COOH terminus. a, shown
is a schematic structure of human La protein. La protein is composed of
408 amino acids. The predicted caspase-3 cleavage site(s) in the COOH
terminus are indicated (underlined). RRM, RNA recognition motif. b,
antisera against the NH
2
- or COOH-terminal peptides (see a, bold lines)
were raised in rabbits. Cell lysates were prepared from untreated (⫺)or
camptothecin-treated (⫹) HL-60 cells and immunoprecipitated with
normal mouse immunoglobulin (normal mu-Ig) or anti-La mAb 24.
Each precipitate was subjected to Western blot analysis with anti-NH
2
-
terminal or anti-COOH-terminal peptide antibody.
FIG.5. Production of a 43-kDa fragment by exogenous
caspase-3 in vitro and effects of caspase inhibitors on cleavage
of La protein. a, cell lysates were prepared from non-apoptotic HL-60
cells, incubated with (⫹) or without (⫺) recombinant caspase-3 at 37 °C
for 2 h, and subjected to Western blotting with anti-La mAb 24. For
comparison with in vitro cleaved product of La protein, cell lysates were
prepared from untreated (control (C)) and camptothecin-treated (CPT)
HL-60 cells. b, HL-60 cells were preincubated with the indicated con-
centrations of the specific inhibitor of caspase-3 (acetyl-DEVD-aldehyde
(DEVD-CHO)) or with the specific inhibitor of caspase-1 (acetyl-YVAD-
aldehyde (YVAD-CHO)) for 1 h, followed by treatment with 1
M camp-
tothecin or 15
M etoposide for 5 h. Cell lysates were subjected to
immunoblotting analysis with anti-La mAb 24.
FIG.6. Measurements of molecular mass of the 43-kDa frag-
ment. To map the cleavage site, La protein and the 43-kDa fragment
were isolated from etoposide-treated HL-60 cells by anti-La mAb affin-
ity chromatography and subjected to DE-MALDI-TOF-MS (6.5-m flight
length). The 43-kDa fragment was detected as a single peak with a
mass of 43,182 ⫾ 44 Da (n ⫽ 5). The mass of the native La protein is
46,831 ⫾ 31 Da (n ⫽ 5). Two minor unidentified peaks were also seen.
Apoptosis-associated Cleavage and Redistribution of La Proteins34468
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suggest that loss of NLS may be responsible for the redistribu-
tion of La protein to the cytoplasm during apoptosis.
DISCUSSION
Our results demonstrate that La protein loses its COOH-
terminal NLS by proteolytic cleavage during apoptosis and that
the loss of NLS leads to the redistribution of La protein from
the nucleus to the cytoplasm. Apoptosis is accompanied by
disorganization of the nuclear architecture, cytoskeleton, and
cell membrane. Members of the caspase family of cysteine
proteases play essential roles in apoptosis (1–3). The caspase
proteases have overlapping substrate specificities. For exam-
ple, caspase-2, caspase-3, and caspase-7 each display similar
specificities, which suggests that their roles in cells are at least
overlapping, if not completely redundant (33, 34). The DEXD
motif is present in the cleavage sites of several cell mainte-
nance and/or repair proteins that are proteolytically cleaved
during apoptosis, including poly(ADP-ribose) polymerase (32–
34), the catalytic subunit of DNA-dependent protein kinase
(28), the 70-kDa protein component of the U1 small ribonucleo-
protein (28), D4-GDI (38), and protein kinase C
␦
(39).
Amino acid sequence analysis of the COOH-terminal portion
of human La protein revealed a DEHD sequence repeat that
fits the DXXD motif,
368
DEHDEHD
374
. Thus, there are two
possible cleavage sites for caspase-3, Asp-371 and Asp-374; but
La protein is cleaved only at Asp-374 in apoptotic HL-60 cells
upon apoptosis. This result demonstrates that the cleavage site
in apoptotic cells is not solely determined by the DXXD motif,
but that other factors such as the amino acid sequence sur-
rounding the DXXD motif, interaction with other molecules, or
conformation may influence the acceptability of each DXXD
sequence.
La protein is cleaved less efficiently than the well docu-
mented caspase-3 substrate of PARP, indicating that the cleav-
age site of La protein is less accessible to caspases than that of
PARP. After submission of this manuscript, Rutjes et al. (40)
reported that La protein was proteolytically cleaved in vivo,
generating a 45-kDa fragment. Based on the molecular size of
the fragment upon SDS-polyacrylamide gel electrophoresis and
the observation that caspase-specific inhibitors prevented
cleavage of La protein, they proposed that La protein might be
cleaved at Asp-371 and/or Asp-374 upon apoptosis. Their re-
sults are consistent with ours.
Sera from patients with rheumatoid diseases such as sys-
temic lupus erythematosus and Sjo¨gren’s syndrome frequently
contain antibodies directed against La (18, 19). The mecha-
nisms responsible for production of autoantibodies against La
protein are not fully understood. As shown in this study, La
protein shows distinctive behavior during apoptosis, and this
may be a general phenomenon associated with apoptosis of
various types of cells. Apoptotic cell antigens have been iden-
tified as the targets of autoantibodies in autoimmune diseases,
and it was proposed that apoptotic cells were the primary
source of autoantigens (20, 22).
Casciola-Rosen et al. (20) showed that La antigen was found
to rim the core of nucleic acid in early apoptosis and then was
found surrounding apoptotic bodies (large blebs) at the cell
surface in later stages of apoptosis in UV-irradiated keratino-
cytes. Miranda et al. (21) reported that nuclear La staining
became weaker and that La-stained blebs emerged from the
cell membrane in later stages of apoptosis in cardiac myocytes.
It would be interesting to determine whether loss of NLS by
caspases is responsible for the redistribution of La protein in all
of these cases.
Acknowledgments—We thank Drs. M. Shiohara (Shinshu University
School of Medicine), M. Nakajima (Novartis Pharma Co., LTD), and T.
Yamori (Japanese Foundation for Cancer Research) for helpful com-
ments regarding this manuscript.
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Aoyama and Junji Sagara
Haritha Sarvotham, Astushi Hara, Toshifumi
Junya Masumoto, Shigenari Hashimoto,
Koichi Ayukawa, Shun'ichiro Taniguchi,
Localization Signal during Apoptosis
Terminus and Loses the Nuclear
La Autoantigen Is Cleaved in the COOH
DEVELOPMENTAL BIOLOGY:
MOLECULAR BASIS OF CELL AND
doi: 10.1074/jbc.M003673200 originally published online July 26, 2000
2000, 275:34465-34470.J. Biol. Chem.
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