From the Department of Radiation Oncology, Laboratory of Molecular Stress Responses, Ireland Comprehensive Cancer Center and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942
Received for publication, September 9, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Nuclear clusterin (nCLU) is an ionizing radiation
(IR)-inducible protein that binds Ku70, and triggers apoptosis when
overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU
synthesis is a product of alternative splicing. Reverse
transcriptase-PCR analyses revealed that exon II, containing the first
AUG and encoding the endoplasmic reticulum-targeting peptide, was
omitted. Exons I and III are spliced together placing a downstream AUG
in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU.
Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU
interaction. The N- and C-terminal coiled-coil domains of nCLU
interacted with each other, suggesting that the protein could dimerize
or fold. Mutation analyses indicate that the C-terminal NLS was
functional in nCLU with the same contribution from N-terminal NLS. The
C-terminal coiled-coil domain of nCLU was the minimal region required
for Ku binding and apoptosis. MCF-7 cells show nuclear as well as
cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic
aggregation of GFP-nCLU was found in viable cells. These results
indicate that an inactive precursor of nCLU exists in the cytoplasm of
non-irradiated MCF-7 cells, translocates into the nucleus following IR,
and induces apoptosis.
The clusterin (CLU)1
gene was originally isolated by numerous laboratories due to its
up-regulation during cell death responses in various tissues and after
various toxic stress signals (1-11). CLU expression is complex,
appearing as different forms in different cell compartments. One set of
proteins is directed for secretion, and other CLU species are expressed
in the cytoplasm and nucleus (see below). The secretory form of the
clusterin protein (sCLU) has been extensively studied and is produced
by translation from the first AUG codon of the full-length CLU
mRNA. sCLU is targeted to the ER by an initial leader peptide. This
~60-kDa pre-sCLU protein is further glycosylated and proteolytically
cleaved into The role(s) of CLU in cell death processes is also complex and may be
different for the various forms of the protein. Recent data suggest
that overexpression of sCLU in human cancer cells caused drug
resistance and protection against certain cytotoxic agents that induce
apoptosis (9, 15, 16). Additional confirmatory data suggest that sCLU
acts as a molecular chaperone, scavenging denatured proteins outside
cells following specific stress-induced injury such as heat shock
(17-21). In fact, sCLU possesses nonspecific binding activity to
hydrophobic domains of various proteins in vitro (20),
further supporting its role as a molecular chaperone acting to clear
cellular debris. sCLU does not associate with Ku70 (14).
Other data, including our own (13), show that overexpression of a
specific nuclear form of CLU (nCLU) acts as a pro-death signal,
inhibiting cell growth and survival. Our laboratory isolated CLU while
searching for Ku70-binding proteins using a yeast two-hybrid screen of
a human liver cDNA library (14). The specificity of nCLU-Ku70
interaction was confirmed by co-immunoprecipitation, far Western, and
confocal co-localization analyses of the two proteins in transfected or
IR-treated MCF-7:WS8 (MCF-7) breast adenocarcinoma cells (13). Confocal
microscopy revealed an apparently inactive form of nCLU
(i.e. pnCLU) in the cytoplasm of non-irradiated cells (13)
that subsequently translocated to the nucleus following clinically
relevant low dose IR exposures ( Interestingly, an ~49-kDa nuclear CLU precursor (pnCLU) was induced
and translocated from the cytoplasm to the nucleus after certain
cytotoxic events, including IR (13) or transforming growth factor- Analyses of the amino acid sequence of full-length rat CLU detected
four "myosin tail-like" domains (24) that are synonymous with a
"coiled-coil" structure. Coiled-coil domains are the most common
motifs used by proteins for oligomerization, and consist of amphipathic
The objectives of the following studies were as follows: (a)
investigate the mechanism(s) of nCLU production; (b)
identify the functional NLS of the protein; (c) identify the
death domain of nCLU, and (d) elucidate the
Ku70-binding domain of nCLU. These studies may then allow elucidation
of the role of nCLU-Ku70 binding in cell death responses. We
demonstrate that nCLU has one high affinity coiled-coil Ku70 binding
domain within its C terminus. This domain is essential for its cell
death function. Furthermore, nCLU contains an additional coiled-coil
domain in its N terminus that interacts with its own C-terminal
coiled-coil domain. Interestingly, the N-terminal coiled-coil domain of
nCLU does not interact with Ku70. Furthermore, an N-terminal nCLU
coiled-coil-containing peptide interfered with binding of the nCLU
C-terminal coiled-coil domain with Ku70. Based on the apparent physical
interactions of N- and C-terminal pnCLU coiled-coil domains, we offer a
mechanism by which the inert ~49-kDa pnCLU could become activated to
an ~55-kDa nCLU death protein. Finally, we discuss the cell death
function of nCLU in damaged cells, and we propose mechanisms of cell
death involving Ku70 binding.
RT-PCR of nCLU--
Based on probable splice sites, the
following primers were used to investigate whether alternative
splice forms of CLU could be found: hCLU5', 5'-ACAGGGTGCCGCTGACC-3';
hCLUN-term-rev, 5'-TTAGAGCTCCTTCAGCTTTGTCTC TG-3'; NCLUforw-3,
5'-GCTGACCGAAATGTC-3'; hnCLUrev, 5'-TCACACCACCCGGTGCTTTTTG-3'. Total RNA from MCF-7 cells was extracted using RNAsol B
(Invitrogen). Reverse transcription reactions were performed using the
oligo(dT) primer (Promega) and Superscript II enzyme system
(Invitrogen). The PCR step was performed using Taq DNA
polymerase (Invitrogen). When hCLU5' and hCLUN-term-rev primers were
used, PCR conditions were as follows: 95 °C for 30 s,
62-72 °C for 45 s, and 72 °C for 45 s. When NCLUforw-3
primer was used in pair with either hCLUN-term-rev or hnCLUrev primers,
optimal conditions were as follows: 95 °C for 30 s, 64 °C
for 45 s, and 72 °C for 45 s. A Bio-Rad iCycler Thermal
Cycler was used for all reactions. RT-PCR products were resolved by 2%
agarose gel electrophoresis and purified using the Concept Matrix Gel
Extraction System (Invitrogen). DNA sequencing was performed by the
Case Western Reserve University DNA Sequencing Service.
Computer Analyses of Protein and Nucleic Acid Primary and
Secondary Structures--
EMBnet software
(www.ch.embnet.org/software/COILS_form.html) was used to analyze amino
acid (aa) sequences of mouse and human CLU proteins for coiled-coil
domain structures; coiled-coil domains in proteins are known to be
important for protein-protein interactions (25-27). Analyses were
performed using the algorithm published by Lupas et al.
(28). Primary sequences of both human and mouse CLU proteins were
retrieved from GenBankTM (accession numbers: S70244 for
mouse and M64722 for human CLUs (12, 29)). Construction of PCR primer
sets for deletion/mutation analyses of the CLU protein was performed
using OligoTech version 1.00 software (Oligos Etc., Inc./Oligos
Therapeutics, Wilsonville, OR).
Generation of CLU Mutants and Yeast Two-hybrid Analyses--
The
Y190 yeast strain (HIS3,lacZ,trp1,leu2 (30, 31)) was used in
two-hybrid structure/function analyses as described (13, 14).
Full-length mouse CLU cDNA was a generous gift from Dr. M. Tenniswood (Notre Dame University). Full-length mouse Ku70 cDNA was
a generous gift from Dr. M. Abe (National Institute of Radiological
Sciences, Japan). Human CLU and Ku70 constructs were used as described
previously (14). Mouse CLU deletion mutants, containing aa residues
18-448, 18-97, 98-317, 18-380, 18-328, 256-328, 345-380, and
318-368, and human CLU deletion fragments 34-449 and 245-380 were
created by PCR using the appropriate primer sets (described above).
Deletion mutants were inserted into either pAS2-1 or pACT2 vectors
(Clontech Laboratories, Inc., Palo Alto, CA) as
prey or bait, respectively, using SmaI sites and the
Perfectly BluntTM Cloning kit (Novagen, Inc., Madison, WI),
according to manufacturer's instructions. Mouse CLU deletion mutants
containing amino acids 256-448, 256-380, and 256-344 were created
using NcoI restriction enzyme digestion in combination with
BamHI, ScaI, or PstI, respectively. The resulting fragments were then inserted into the pAS2-1 vector using
NcoI, BamHI, SmaI, and PstI
restriction sites. Mouse Ku70 cDNA was excised from the original
vector using BspLU11I and EcoRI enzymes, and
inserted into pACT2 and pAS2-1 vectors, which were pre-digested with
NcoI and EcoRI restriction enzymes. Point
mutations within mouse CLU cDNAs were introduced using the
QuikChange kit (Stratagene, Cedar Creek, TX) following the
manufacturer's instructions. The LR mutant (substitution of 9 Leu
residues to Arg within the C-terminal coiled-coil domain of mouse nCLU)
was generated by direct cloning of a 155-bp DNA fragment containing all
required nucleotide substitutions. This was accomplished by first
synthesizing a single strand DNA fragment: 5' GC CAG GAG CGG AAC GAC
TCG CGC CAG GTG GCC GAG AGG CGGACA GAG CAG TAC AAG GAG CGG CGG CAG TCC TTC CAG TCG AAGATG CGC AAC ACC TCA TCC CGG CGG GAG CAG CGG AAC GAC
CAGTTC AAC TGG GTG TCC CAG CGG GCT AAC CGC A 3'. This fragment was
further amplified by PCR using Pfu DNA polymerase
(Stratagene, Cedar Creek, TX) and the following pair of primers:
AlmutPCR1 (30-mer), 5' GCT AAC CTG CGC CAG GAG CGG AAC GAC TCG 3', and
AlmutPCR2 (38-mer), 5' TTA GTA GTA CTT GTC TTC TCC CTG TGT GCG GTT AGC
CC 3'.
The PCR product was gel-purified, digested with the EcoNI
restriction enzyme, and inserted into the pASnCLU,
pAS-(256-448) and pGFPnCLU nCLU vectors, which were
pre-digested with EcoNI to create NCLU-LR, NcoLR,
and GFP-NCLU-LR, respectively. In addition, the undigested PCR product
was inserted into a pcDNA3.1/NT-GFP-TOPO mammalian expression
vector (Invitrogen) using blunt ligation to create the GFP-CcLR mutant.
Human CLU cDNA fragments encoding aa residues 84-449 and 329-449
in the pACT2 vector, as well as human Ku70 cDNA in the pAS2 vector,
were described previously (13, 14). All constructs were sequenced to
verify in-frame positions with respect to the GAL4 domain. GAL4-Ku70
was co-transfected with various GAL4-CLU fragments into Y190 yeast
using the protocol provided in the MATCHMAKER (Clontech Laboratories, Inc., Palo Alto, CA)
manual. Stable transformants were grown for 6 days at 30 °C on
plates containing His( Western Blot Analyses--
Rabbit polyclonal H-330 antibody
(Santa Cruz Biotechnology) was used to detect the 49-kDa species of CLU
(pnCLU) in whole cell extracts from MCF-7 cells. A 1:2500 dilution of
primary H-330 antibody was used followed by 1:10,000 dilution of
horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa
Cruz Biotechnology).
Yeast extracts were prepared using acid-washed glass beads as described
in the MATCHMAKER kit manual and were used for Western blot analyses.
Protein concentrations were determined using the Bio-Rad protein
concentration reagent as per manufacturer's instructions (Bio-Rad).
Yeast extracts were mixed with an equal volume of 2× Laemmli buffer,
boiled for 5 min, and resolved on either 10 or 15% PAGE gels,
depending on the expected molecular weight of the proteins of interest.
Proteins were then transferred onto polyvinylidene difluoride-Plus
membranes (MSI, Westboro, MA). Membranes were blocked with 5% non-fat
dried milk and probed with anti-GAL4BD or anti-GAL4AD monoclonal
antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Horseradish peroxidase-conjugated anti-mouse secondary antibody
(Amersham Biosciences) was then used, and proteins were visualized by
ECL (Amersham Biosciences) and x-ray film. All CLU deletion or point
mutants were approximately equally expressed in yeast, and no loss of
stability of proteins was noted.
In Vitro GST-Ku70 Binding Assays--
Full-length mouse Ku70
cDNA was amplified using PCR and the appropriate primer sets and
inserted into the pGEX-2T vector (Amersham Biosciences) at a
SmaI restriction site to generate an in-frame glutathione
S-transferase (GST)-fused protein. Escherichia
coli NovaBlue Single Competent Cells (Novagen, Inc., Madison, WI)
were used for plasmid propagation according to the protocol described in the Perfectly BluntTM Cloning kit manual (Novagen, Inc.,
Madison, WI). GST or GST-Ku70 fusion proteins were generated and
purified using standard methods (32). All genetically manipulated
plasmids were confirmed by DNA sequencing prior to use.
Glutathione-agarose beads (Amersham Biosciences) were used for
purification of GST-fused proteins as described (32) using Binding
buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl,
1.5 g/liter bovine serum albumin, proteinase inhibitors). Amounts of
immobilized GST-Ku70 or GST (alone) proteins were estimated by
Coomassie Blue staining of SDS-PAGE gels, with various amounts of
bovine serum albumin protein used as standards. Mouse CLU cDNA fragments encoding amino acid residues 18-448 and 345-380 were amplified by PCR and inserted into the pT7Blue-2 vector (Novagen, Inc.,
Madison, WI) using the Perfectly BluntTM Cloning kit
(Novagen, Inc., Madison, WI) according to manufacturer's instructions.
All cDNA expression vectors were sequenced to confirm cloning
before use.
The TNT-coupled wheat germ extract system (Promega,
Madison, WI) was used to generate S·Tag-fused,
35S-labeled CLU protein fragments in vitro
according to manufacturer's protocols. The amount of
35S-labeled protein was estimated using the S·Tag Rapid
Assay kit (Novagen Inc., Madison, WI), and an equal amount of
transcription/translation mixture (40 µl) was added to
glutathione-containing beads at the same molar amounts (0.2 pmol of CLU
to 0.2 pmol of GST or GST-Ku70) and incubated overnight at room
temperature. The mixture was then incubated at 4, 23, or 37 °C for
1 h, and beads were collected by centrifugation (15,000 × g), washed three times with ice-cold Binding buffer,
resuspended in Laemmli buffer, and boiled, and proteins were resolved
by 10-15% SDS-PAGE. 35S-Labeled nCLU fragments were then
visualized by autoradiography as described (33).
Intracellular Trafficking of nCLU and Mutant nCLU
Proteins--
To study the intracellular targeting of various nCLU
fragments, we transfected MCF-7 cells with GFP-nCLU and GFP-nCLU
deletion mutants. Effectin reagent (Qiagen Inc., Valencia, CA) was used to transfect 2 × 104 log phase MCF-7 cells that were
pre-attached for 24 h to coverslips. Forty eight hours (48 h)
later, cells were fixed for 1 h in freshly prepared 2%
paraformaldehyde at room temperature. Cells were then rinsed twice in
phosphate-buffered saline and treated with RNase A in
phosphate-buffered saline (1 µg/ml) for 20 min at 37 °C. Cells
were mounted in propidium iodide-containing Vectashield mounting media
(Vector Laboratories Inc., Burlingame, CA) and subjected to confocal
microscopy using a Bio-Rad MRC 1024 Confocal Laser Scanning microscope
(Bio-Rad). All photomicrographs show representative z-sections at
×1000 magnification. At least 180 transfected cells (green) were
visually screened for distribution of GFP-CLU protein in the cell and
its co-localization with propidium iodide-stained nuclei (red). The
number of cells with pycnotic propidium iodide versus normal
nuclear morphology was manually counted, and percentages of apoptotic
cells ± S.E. were calculated (see Table I). Likewise, the number
of cells with nuclear only, cytoplasmic and nuclear, or cytoplasmic
only localization of the GFP-fused proteins was manually counted.
Percentages of cells with nuclear (or cytoplasmic and nuclear) or
cytoplasmic only expression ± S.E. were calculated (Table
II).
Identification of an nCLU-specific mRNA Splice
Variant--
After examining exon/intron sequences of the human
CLU gene, we noticed an acceptor splice site two
nucleotides upstream from the second in-frame AUG (codon
34) sequence of pnCLU (Figs. 1 and
2A). Located within exon III,
this AUG codon was proposed to be the start site for the intracellular
pnCLU protein (13, 22). In contrast to previous alternative translation
start theories, we hypothesized that nCLU could be translated from low
abundance mRNA produced by an alternative splicing event when exons
I and III are spliced together. In this case, exon II that contained the first AUG and the ER-targeting signal of sCLU would be omitted. To
verify this hypothesis, we used specifically designed primers, hCLU5'
to exon I and hCLUN-term-rev to exon IV (see "Experimental Procedures"), to amplify the 5' ends of various splice variants of
CLU cDNA derived from total RNA extracted from MCF-7 cells. The
resulting RT-PCR fragments are shown in Fig. 2B. All visible fragments were excised from the agarose gel and sequenced. As expected,
the 345-bp major fragment was identical in sequence to the 5' end of
sCLU. Two minor fragments with sizes of 450 and 550 bp had additional
exons in the 5'-untranslated region and, therefore, did not contain any
alterations in the open reading frame. Sequencing of the 220-bp
fragment demonstrated, as we predicted for nCLU mRNA, the lack of
exon II and direct junction between exons I and III (see Fig.
2C, sequence).
By using the primer to the 3' end of the CLU open reading frame, we
amplified full-length nCLU cDNA from MCF-7 cells. Sequence analyses
confirmed the expected alternatively spliced full-length nCLU mRNA.
No additional changes in the downstream exon composition of the nCLU
cDNA were found.3 Based
on the data above, we concluded that human nCLU protein was translated
from a separate, minor species of mRNA lacking exon II as a result
of an alternative splicing event.
Identification and Analyses of Potential Functional Domains in
nCLU--
Analyses of the proposed primary aa composition of human and
mouse CLU proteins, based upon respective CLU mRNA sequences, revealed two potential coiled-coil domains (Fig. 1). One coiled-coil domain was located in the N terminus (aa residues 42-98 in human and
42-97 in mouse) of nCLU, and the other coiled-coil domain was located
in the C-terminal portion of both human and mouse nCLU proteins
(residues 319-349 in human and 318-366 in mouse). Both C- and
N-terminal domains appeared to be highly conserved and corresponded to
"myosin-like" domains I and IV, identified previously by Tsuruta
et al. (24). Both N- and C-terminal coiled-coil domains of
CLU were rich in leucines and charged aa residues. We compared the
characteristic 4-3 repeats of known coiled-coil domain-containing
proteins GCN4, c-Jun, c-Fos, and BZLF1 (34-38) to the N- and C-terminal
coiled-coil domains of human and mouse nCLU (Fig. 4A). The
C-terminal coiled-coil domain of nCLU contains a 4-3 repeat of
predominantly hydrophobic residues, much like the BZLF1 gene product of
the Epstein-Barr virus (38). The C-terminal coiled-coil domain of nCLU
did not, however, contain a classical leucine zipper heptad. The 4-3 pattern within the predicted N-terminal coiled-coil domain
consisted of both hydrophobic and charged residues.
Sequence analyses also indicated that the N-terminal coiled-coil domain
of nCLU contained an SV40 large T-antigen-like NLS, LEEAKKKK (residues
74-81 in human and 73-80 in mouse) (22). In addition, we identified
two other potential NLSs. One NLS-like sequence was found only in human
nCLU (RRELDESLQVAERLTRK, residues 324-340) and was similar to the
bipartite NLS found in the Xenopus nucleoplasmin protein
(39). This potential NLS was not found in rodent nCLU protein. The
other potential NLS in nCLU, RKKHR (residues 442-446) in human and
RRKSR (residues 442-446) in mouse, was located at the very C terminus
of the nCLU protein. Because functional NLSs in nCLU have also not been
elucidated, we examined the role of coiled-coil domains and NLS sites
for the cell death function of the protein and for association with Ku70.
The C-terminal Coiled-coil Domain of nCLU Is Essential for Binding
to Ku70--
A number of deletion mutants of human and mouse nCLU were
constructed and tested for Ku70 binding using yeast two-hybrid analyses (Fig. 3A). Only those mutants
containing the C-terminal coiled-coil domain, or a portion of the
domain, were capable of interacting with the Ku70 protein. nCLU
polypeptides containing the N-terminal domain (residues 18-97), a
central region (residues 98-317), or a C-terminal fragment
(residues 381-448) of the protein did not interact with Ku70. None of
these nCLU fragments contained the C-terminal coiled-coil domain. Two
nCLU polypeptides encompassing aa residues 256-344 and 345-380,
respectively, contained only a portion of the C-terminal coiled-coil
domain. Interestingly, these two nCLU fragments showed strong
interaction with Ku70. Steady-state expression of GAL4-fused proteins,
both Ku70-binding and non-binding polypeptides, are shown in Figs.
3B and 4B. These findings suggested that the C-terminal coiled-coil domain of nCLU was
important for its interaction with Ku70. Furthermore, these data
suggested that half, or less, of the coiled-coil domain was sufficient
to bind Ku70 in yeast two-hybrid assays.
To study the role of specific residues in nCLU-Ku70 binding, we
concentrated on the nCLU polypeptide fragment containing residues 345-380 (nCLU-(345-380)). This fragment displayed strong
binding to Ku70 and contained only 22 residues of the predicted
C-terminal coiled-coil domain. Because the Ku70-binding domain of nCLU
contained a putative 4-3 hydrophobic repeat motif, we determined
whether specific leucine repeat residues within nCLU-(345-380) were
necessary and sufficient to mediate Ku70-nCLU binding. The following
point mutations were introduced: (a) L357R,L358Q (a
double substitution of leucines 357 and 358 to arginine and glutamine,
respectively); (b) E359K; (c) L361R;
(d) D363H; (e) L371R; and (f) L374R,
L357R,L358Q, E359K, L361R, and D363H were point mutations made inside
the nCLU coiled-coil domain, whereas L371R and L374R were substitutions made outside the coiled-coil domain (Fig. 4B). Substitution
of leucine residues inside the coiled-coil domain structure of
nCLU-(345-380) completely abrogated Ku70 binding. Substitutions of
leucine residues outside the coiled-coil domain decreased the
association of each nCLU-(345-380) peptide with Ku70 but did not
completely abrogate protein-protein interaction. Mutations of two
non-leucine residues that were not part of the 4-3 repeat within the
same region of the protein (E359K and D363H) did not affect
nCLU-(345-380) binding to Ku70. These data strongly suggested that
only residues that constituted the 4-3 hydrophobic repeat inside the
C-terminal coiled-coil domain of nCLU mediated its interaction with
Ku70. Leucines 371 and 374 immediately outside the coiled-coil domain
of nCLU may, therefore, stabilize protein-protein interaction but do
not appear to be essential for Ku70 binding.
Additional evidence suggesting the importance of the coiled-coil
structure of nCLU and its interaction with Ku70 came from deletion
analyses using human nCLU cDNA/protein and human Ku70 as bait in
yeast two-hybrid analyses (Fig. 3A). Our limited analyses suggest that aa residues 326-365 are minimally essential for the association of nCLU with Ku70. The human homolog portion of the mouse
nCLU-(345-380) polypeptide fragment was unable to bind human Ku70
(Fig. 3A). This fragment of human nCLU contained leucines in
positions 357, 358, and 361 that were essential for mouse nCLU binding
to Ku70. However, the predicted C-terminal coiled-coil domain of human
nCLU was shorter than in mouse nCLU. This shortened length of the
C-terminal coiled-coil domain of human nCLU was apparently due to the
presence of a tryptophan residue at position 350 in human nCLU that
presumably interferes with
To confirm the role of hydrophobic residues in the context of
full-length nCLU for Ku70 binding, we created mouse nCLU mutants (designated as LR mutants) with nine leucine residues within the C-terminal coiled-coil domain substituted by arginines as described under "Experimental Procedures." Leucines 326, 330, 336, 343, 344, 352, 357, 358, and 361 were mutated to arginines (LR mutant) in the
full-length nCLU (nCLU-LR), nCLU-(256-448) (NcoLR), as well
as nCLU-(218-365) (CcLR). NCLU-LR and NcoLR mutants were tested by yeast two-hybrid analyses for Ku70 binding, along with their
wild-type counterparts. Both NCLU-LR and NcoLR mutants were unable to bind mouse Ku70 protein (Fig. 4D) under conditions
where each mutant protein was expressed in transformed yeast cells
(Fig. 4E). In contrast, their wild-type counterparts (nCLU
and nCLU 256-448) strongly associated with Ku70.
To investigate the role of the GST-Ku70 Pull-down Analyses Confirm Yeast Two-hybrid
Results--
To confirm the yeast two-hybrid data, we utilized
GST-Ku70 in vitro binding (pull-down) assays. GST-Ku70 and
GST proteins were expressed in bacteria and immobilized on
glutathione-Sepharose beads as described under "Experimental
Procedures" (Fig. 5B). 35S-Labeled mouse nCLU polypeptide was used to determine
optimal Ku70-binding conditions (Fig. 5A). Minimal nCLU
binding to GST was detected in a temperature-insensitive fashion as
described by Humphreys et al. (20). Significant interaction
of nCLU with GST-Ku70 was detected when incubation temperatures were
between 23 and 37 °C, whereas nCLU binding to an equal amount of GST
alone did not change with temperature (Fig. 5A). At 4 °C,
nCLU binding to GST-Ku70 was not significantly higher than background.
These data indicated a temperature-dependent association of
nCLU binding to Ku70, and that a temperature-sensitive conformational
change was necessary for protein-protein association.
Point mutations in mouse nCLU fragment 345-380 at L357R,L358Q, E359K,
L361R, D363H, and L371R were then examined using GST-Ku70 in
vitro binding assays (Fig. 5C). The results were
identical to those obtained by yeast two-hybrid analyses, except that
mutant E359K showed unexpectedly high binding to GST-Ku70 compared with wild-type nCLU, suggesting that some non-4-3 repeat residues inside the
coiled-coil domain might also modulate Ku70 binding.
The Coiled-coil Domains of nCLU Bind Each Other--
The longest
Ku70-binding nCLU polypeptide (residues 84-449), isolated from the
human liver cDNA library by yeast two-hybrid screening, excluded
most of the N-terminal coiled-coil domain (14). However, all nCLU
polypeptides that interacted with Ku70 contained the C-terminal
coiled-coil domain. Interestingly, full-length nCLU cDNA containing
C- and N-terminal coiled-coil domains was not isolated in the initial
yeast two-hybrid screen (14). The reasons for the absence of
full-length nCLU were unclear. One possible explanation for the lack of
isolation of full-length nCLU cDNA was that the N-terminal
coiled-coil domain might bind to the C-terminal coiled-coil domain of
nCLU and interfere with Ku70 binding (see below). Yeast two-hybrid
analyses of a number of nCLU deletion mutants showed that whenever two
coiled-coil domains of nCLU were present in the same molecule
(e.g. polypeptides containing aa residues 18-380 of mouse
nCLU and human nCLU fragment containing aa residues 34-449), binding
of these fragments to Ku70 was notably lower compared with nCLU
polypeptides containing a single C-terminal coiled-coil domain
(i.e. see mouse nCLU mutants containing residues 256-448,
and the human nCLU polypeptide fragment containing residues 84-449)
(Fig. 3A). These data suggested to us that the N-terminal
coiled-coil domain of nCLU might compete with Ku70 for binding to the
C-terminal coiled-coil domain of nCLU.
Because the N-terminal coiled-coil domain of nCLU did not interact with
Ku70 (Fig. 3A), we hypothesized that this N-terminal domain
of nCLU might act as a suppressor of nCLU-Ku70 protein-protein interaction. Therefore, we examined the ability of coiled-coil domains
of nCLU to bind each other. The C-terminal coiled-coil domain of human
nCLU (i.e. a fragment containing aa residues 329-449) was
used as "bait." The C-terminal coiled-coil domain of nCLU was also
able to interact with the N-terminal domain of the protein (aa 18-97)
and with itself (aa 256-380) (Fig.
6A). However, neither the
central portion of nCLU (aa 98-317) nor a C-terminal fragment of nCLU
missing the coiled-coil domain (aa 381-448) interacted with either the
C- or N-terminal coiled-coil domains of nCLU. In addition, when tested
as bait, the N-terminal coiled-coil domain interacted only with the
C-terminal coiled-coil domain (Fig. 6B). The N-terminal
coiled-coil domain did not interact with itself nor with central or
C-terminal fragments of nCLU that lacked coiled-coil structures. These
data strongly support the theory that the coiled-coil domains of nCLU
may indeed be involved in either intra- or intermolecular interactions.
Intracellular Targeting of nCLU Domains and Induction of Apoptosis
by Specific nCLU Proteins--
Previous analyses of the nCLU aa
sequence identified one NLS within its N-terminal coiled-coil domain
(40) and another within the C-terminal fragment, but outside its
coiled-coil domain (13). To investigate the role of these NLSs in the
subcellular localization of the nCLU protein and in the cell death
functions of the protein, we transfected MCF-7 breast cancer cells with
GFP-fused constructs of wild-type and NLS mutants of the nCLU protein
(used above). MCF-7 cells were chosen because we characterized
previously (13) nCLU translocation in these cells following IR.
Confocal microscopic analyses of the subcellular localization of
various GFP-CLU fragments in MCF-7 cells 48 h post-transfection
were then noted (Fig. 7), and the
percentage of apoptosis ± S.E. was quantified (Table
I). Surprisingly, exogenously
overexpressed full-length GFP-nCLU fusion protein was mostly cytosolic
with minor expression noted in the nuclei of some transfected
(green) cells (Fig. 7, B and C). In fact, the GFP-nCLU-positive population could be divided into two types.
One population contained cells with normal nuclear morphology and very
bright GFP-nCLU aggregates in the cytosol and peri-nuclear space (Fig.
7C), similar to untreated MCF-7 cells stained for endogenous
nCLU protein (13). These aggregates did not co-localize with the ER,
the Golgi apparatus, or mitochondria when co-stained with the following
organelle-specific fluorescent markers: Texas Red-conjugated
concanavalin A, Texas Red X-conjugated wheat germ agglutinin), and
MitoTracker Red (Molecular Probes, Eugene, OR). Virtually no GFP-nCLU
was detected in nuclei of cells within this subpopulation of
transfectants, and virtually none of these cells showed classical
apoptotic morphology. Another subpopulation of GFP-nCLU transfectants
(~15% of GFP-nCLU-positive cells as determined by visual screening)
had diffuse distribution of GFP-nCLU with some apparent GFP-nCLU
expression in nuclei (Fig. 7B). Most importantly, all cells
that had visible nuclear GFP-nCLU expression had condensed chromatin,
characteristic of apoptotic cells (41). Expression of GFP-Nterm protein
in MCF-7 cells was diffusely distributed and located in both the
cytosol and nucleus of transfected cells (Fig. 7D). In
contrast, expression of the GFP-Center fusion protein within MCF-7
cells resulted in the formation of cytosolic aggregates (Fig.
7E), similar to the pattern of protein expression observed in the non-apoptotic subpopulation of the GFP-nCLU MCF-7 transfectants, as well as to the endogenous nCLU expression pattern in non-irradiated MCF-7 cells (13). Few, if any, GFP-Center-transfected MCF-7 cells
exhibited apoptosis or any other abnormal morphology. These data
strongly suggested that the apoptotic effects of nCLU are specific and
that cytosolic localization and aggregate formation are attributed to
protein regions separate from the domain responsible for nCLU-induced
cell death. In contrast, the distribution of GFP-Ccoil (Fig.
7F) protein in both cytosol and nucleus resembled the
subpopulation of apoptotic cells transfected with GFP-nCLU (Fig.
7B). As with GFP-nCLU, the presence of apoptotic nuclei following GFP-Ccoil transfection coincided with GFP-positive nuclear staining, and few apoptotic, non-transfected cells were noted within
the transient cell population. No GFP-positive cells with large
aggregates were detected in the GFP-Ccoil transfectants, as observed in
the non-apoptotic GFP-positive subpopulation present within 48 h
following GFP-nCLU transfection (Fig. 7C). Transfection of
MCF-7 cells with GFP-End (that lacks a coiled-coil domain but has a
C-terminal NLS) resulted in very few cells with large aggregates in
their cytoplasm, and the majority of protein was expressed in the
nuclei of GFP-positive MCF-7 cells (Fig. 7G). Unlike
transfection with GFP-nCLU (Fig. 7B and Table I), few cells
with apoptotic morphology were detected among the GFP-End
transfectants. A single L343P mutation within the C-terminal
coiled-coil domain of GFP-nCLU (Fig. 7H, Table I) resulted
in little or no detectable cell death and punctated cytoplasmic
localization of the GFP-nCLU L343P protein. Transfection of MCF-7 cells
with vector expressing GFP alone resulted in cells with diffusely
distributed protein expression, mostly in the cytoplasm of transfected
cells (Fig. 7A). Few apoptotic or other abnormal
morphologies were noted in GFP-positive expressing MCF-7 cells after
transfection with GFP alone, as found with GFP-Nterm, GFP-Center, and
GFP-End (Fig. 7, D, E, and G and Table
I).
nCLU Protein Has Two Functional NLS Motifs--
It was proposed
previously (13, 22) that the nCLU protein contains two conserved
potential NLSs among species. We define the N-terminal NLS as NLS1 and
C-terminal as NLS2. To identify the functional NLS of nCLU, we
introduced point mutations in the respective NLS sequences. We mutated
NLS1, 77KKKK80 to
77KAVK80 in the context of mouse GFP-Nterm
protein (NLS1mut), and NLS2, 442RRKSR446 to
442VVKSR446 in the context of mouse GFP-End
protein (NLS2mut). The nCLU protein fragments were chosen because they
elicited no apoptotic responses, which could influence the trafficking
analyses. We transfected MCF-7 cells with NLS1mut, NLS2mut, GFP-Nterm
wild-type, GFP-End wild-type or GFP alone, and we determined the
intracellular localization of the GFP-fused protein 48 h
post-transfection (Fig. 8). The percentage of cells with cytoplasmic versus nuclear
localization of GFP-fused proteins are summarized in Table
II. Although GFP-alone control cells
expressed GFP predominantly in the cytoplasm (Fig. 8A), we
noticed uniform distribution of the wild-type GFP-Nterm in 57 ± 5% cells with expression in both the cytoplasm and nucleus (Fig.
8B). Mutation in the N-terminal NLS led to an ~2.5-3-fold decrease in percentage of cells with detectable nuclear expression of
GFP-Nterm NLS1mut (Fig. 8C, Table II). As in Fig.
7G, localization of GFP-End was predominantly nuclear
(98 ± 0.2% cells with nuclear expression, Fig. 8D).
In contrast, GFP-End NLS2mut was detected only in the cytoplasm in
78 ± 5% of GFP-positive cells (Fig. 8E). Based on
these data, we conclude that both NLS1 and NLS2 of nCLU are functional;
however, NLS2 is a relatively stronger site, resulting in greater
differences in nuclear localization of GFP-End versus GFP-End NLS2mut.
The exact role(s) of alterations in CLU gene expression before and
after cytotoxic stress remain(s) a mystery. Our data indicate that
human cells synthesize CLU protein forms that lack ER-signaling peptides and are destined for functions in the cytoplasm and nucleus. This is in contrast to the sCLUs that are currently thought to exert
cytoprotective effects on exposed cells as well as effects on
non-damaged bystander cells as discussed previously (42, 43).
Collectively, our data suggest that nCLU contributes to cell death.
Previous data (13, 22) indicated the existence of a mature ~55-kDa
nCLU protein. The nCLU protein did not appear to be either glycosylated
or cleaved at its The data presented in this paper begin to address these processes and
are consistent with the proposed hypothetical model of nCLU synthesis
and cell death functions shown in Fig. 9,
and discussed below. Our data show that a unique, shortened form of the
nCLU mRNA is present at low levels (detected only by RT-PCR) in
undamaged cells, and presumably increases after IR exposure in direct
proportion to accumulation of full-length CLU mRNA. The low
abundance, truncated form of the nCLU mRNA is the result of
alternative splicing that eliminates exon II, including the first
strong AUG start site. Translation from this truncated nCLU mRNA
results in the production of an ~49-kDa nCLU precursor protein (i.e. pnCLU) that is initiated from a second downstream AUG
translational start site (located in exon III), and the protein
produced (pnCLU) lacks the ER signal peptide. The ~49-kDa pnCLU
appears to contain at least two functional NLSs that targeted the
protein to the nucleus. Only overexpression of nCLU proteins that have
C-terminal coiled-coil domains and a functional NLS caused cell death.
The nCLU protein has two coiled-coil domains, one at its N terminus that is unable to bind Ku70, and a C-terminal coiled-coil domain that
is uniquely able to associate with Ku70 and is minimally required for
cell death. The N-terminal coiled-coil domain of nCLU can associate
with the C-terminal coiled-coil domain of the nCLU protein. Specific
mutations in, or deletions of, the C-terminal coiled-coil domain of
nCLU simultaneously abrogated Ku70 binding and prevented cell death,
even though these mutant forms of the nCLU protein accumulated in the
nuclei of transfected MCF-7 cells (Fig. 7). Our data further support a
role of Ku70 binding in the cell death function of mature nCLU. We show
that only nCLU proteins containing both the C-terminal coiled-coil
domain and a functional NLS can kill MCF-7 cells after transfection.
Alterations in, or deletions of, the C-terminal NLS or C-terminal
coiled-coil domain abrogated cell death. However, attempts to date to
show conclusively that Ku70 is necessary for cell death caused by nCLU
have yielded inconclusive results due largely to the enhanced apoptotic
indices of Ku70
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-subunits, held together by disulfide bonds
(12, 13). External sCLU is an 80-kDa protein that appears as an
~40-kDa
- and
-protein smear by SDS-PAGE under reducing
conditions (14).
1Gy). Co-immunoprecipitation studies
demonstrated the presence of the Ku80 protein in the Ku70-nCLU complex,
strongly suggesting that nCLU bound to the Ku70/Ku80 dimer (13).
Overexpression of nCLU also blocked Ku70/Ku80 DNA end binding activity
(14); however, the relationship between nCLU binding to Ku70 and cell
death remained undetermined.
(22) treatments. This "death" form of the CLU protein was proposed
to be synthesized from a second in-frame AUG codon (13, 22), although
mechanisms of production were not elucidated. The pnCLU protein
apparently lacks the ER-targeting leader peptide and does not undergo
/
cleavage or extensive glycosylation, as observed with sCLU
(22). pnCLU functions remain unknown. Following IR treatment of MCF-7
cells, an ~55-kDa nCLU protein was expressed at extremely low levels
in the nuclei of exposed cells (13). Overexpression of GFP-fused nCLU
(initiated from the second AUG start codon in its mRNA) or a
C-terminal 120-amino acid fragment (C-120) in MCF-7 cells resulted in
significant growth inhibition, caspase 3-independent apoptosis, and
lethality (13, 23). All attempts to stably express nCLU resulted in
lethality (13) or clones expressing G418 resistance without
nCLU.2 In contrast,
overexpression of sCLU protein did not affect the survival of MCF-7
cells before or after IR stress (14).
-helices usually with a 4-3 pattern of hydrophobic and hydrophilic
residues repeated in heptads. To date, no structure/function studies of
the CLU protein have been performed, and mechanisms of the production
of nuclear forms of this protein has yet to be determined.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), Trp(
), Leu(
) and SD medium (Difco)
containing 2% agarose and 15 mM 3-amino-1,2,4-triazole. Colonies were subjected to on-filter
-galactosidase assays, with X-gal as a substrate. The reaction was considered positive when colonies turned blue within first 120 min of incubation with X-gal. Negative reactions (i.e. blue colonies were not observed)
were indicative of no association, only when protein expression for each CLU or Ku70 mutant could be demonstrated by Western blot analyses
(see below). Blue colonies that appeared after 120 min were regarded as
false-positive signals. X-gal staining was performed on colony-lift
filters as described (14).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural map of the human CLU protein.
Letters and boxes signify the following:
LP, endoplasmic reticulum-targeting hydrophobic leader
peptide; NLS, nuclear localization signal; X,
/
-cleavage site (aa 205) of cytoplasmic ~60-kDa pre-secreted
clusterin (psCLU); slashed boxes represent N- and C-terminal
coiled-coil leucine zipper-like regions, aa 42-98 and 319-349,
respectively. Rectangles, nCLU,
Nterm, Center, Ccoil, and End
signify the regions of CLU used for the generation of GFP-fused
constructs (see Table I and Fig. 7).
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Fig. 2.
A, two potential translation start sites
of nCLU are located in the different exons. Structure of the 5' end of
sCLU cDNA is shown. Splice sites are indicated as black
triangles. Small arrows show translational start sites of sCLU and
nCLU. Endoplasmic reticulum targeting leader peptide (ER-targeting
signal) and nuclear location signal are shown in boxes.
Long arrows indicate primers used to amplify various spicing
variants of CLU cDNA. B, alternative splicing of the 5'
end of CLU mRNA as detected by RT-PCR. PCR reaction was performed
using hCLU5' and hCLU-Nterm-rev primers (see A). Annealing
temperatures were 72, 68.2, and 62 °C (lanes 2-4,
respectively). M (lane 1), DNA marker. Note the
position of nCLU (220 bp) and sCLU (340 bp) products.
C, 5' end of nCLU cDNA. Sequence of the 5' end of
nCLU cDNA that is the result of an alternative splicing event when
exon II is omitted.
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Fig. 3.
A, the C-terminal coiled-coil domain of
nCLU mediates Ku70 binding. The relative strength of nCLU-Ku70
interaction for both human and mouse pairs of proteins using yeast
two-hybrid analyses are indicated as follows: +++, strong binding,
colonies turned blue within 30 min of incubation with X-gal; ++,
moderate binding, yeast colonies turned blue within 60 min; +, weak
interaction, colonies turned blue within 120 min; , no interaction,
no blue colonies within 120 min of incubation with X-gal. nCLU
constructs marked with asterisks were tested using yeast
two-hybrid assays with GFP moieties on their N-terminal ends. The
fusion of GFP to these proteins does not affect their cell death
functions (13). Coiled-coil domains are shown as slashed
boxes. Numbers represent the respective aa residues.
Experiments were performed three or more times. B,
expression of GAL4-fused proteins in Y190 yeast. Representative Western
blot analyses of the expression of mouse Ku70 protein (left
panel) used in two-hybrid analyses shown in A and
GAL4AD- and GAL4BD-fused mouse nCLU deletion mutants (right
panel) in Y190 yeast. Plus signs indicate
binding of the particular nCLU fragment to mouse Ku70 as described in
A.
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Fig. 4.
A, the 4-3 repeats of hydrophobic
residues within the protein-protein interaction domains of nCLU as
compared with known coiled-coil domain-containing proteins. The 4-3 repeat-containing protein-protein interaction domains of GCN4, c-Jun,
c-Fos, and BZLF1 are aligned according to Ref. 38 with predicted
coiled-coil domains of human and mouse nCLU. The predicted C-terminal
(Ccoil) and N-terminal (Ncoil) of human and mouse
nCLU (hCLU and mCLU, respectively) are aligned
with the potential nCLU-binding region of human and mouse Ku70 proteins
(hKu70 and mKu70, respectively). Every 1st, 3rd,
and 4th residue is capitalized and boxed. Leucine
residues in mCLU Ccoil that were targeted for the LR mutation are
underlined. Leucine 343, which was targeted for the L343P
mutation, is circled. Rectangles in the
bottom indicate positions of -helices 15, 16, and 16' as
noted (45). The arrow indicates the position of the
StuI site, which was used to create a deletion that resulted
in a Ku70 mutant protein unable to bind to nCLU (14).
Numbers refer to the positions of the first and last
residues within the full-length proteins. B, identification
of aa residues of nCLU that mediate Ku70 binding. The relative strength
of various mutant forms of mouse nCLU associating with mouse Ku70 were
tested using yeast two-hybrid analyses as described in Fig. 3 and
"Experimental Procedures." Numbers represent the
respective aa residues of mouse nCLU protein. Amino acid residues
targeted for mutations are marked with asterisks. Residues
to the left of the dotted line are inside the
predicted C-terminal coiled-coil domain of mouse nCLU. Residues to the
right side of the dotted line are outside the
functional C-terminal coiled-coil domain. Relative strength of binding
was scored as described in Fig. 3A, and experiments were performed three or
more times. WT, wild type. C, GAL4-fused mutants
of nCLU-(345-380) fragments are expressed in Y190 yeast. Western blot
analyses demonstrate expression of mouse nCLU point mutants within aa
345-380 and the corresponding human aa 345-380 nCLU fragment in Y190
yeast. The relative strength of Ku70 binding was described in Fig.
3A, and all experiments were performed three or more times.
D, LR mutants (nCLU-LR and NcoLR) of mouse nCLU
and mouse nCLU-(256-448) protein fragment and L343P mutant of mouse
nCLU-(256-448) fragment do not bind to Ku70. Yeast two-hybrid analyses
were performed to determine binding of wild-type, L343P, and LR mutants
of mouse nCLU and nCLU 256-448 proteins to Ku70. Mouse nCLU fragment
(aa 318-365) contains C-terminal coiled-coil domain, and wild-type, LR
mutant, and L343P (with a P) nCLU proteins are also shown.
The relative strength of binding of mouse nCLU LR mutants to mouse Ku70
protein was indicated as described in Fig. 3 and "Experimental
Procedures." The experiments were performed three or more times.
E, steady-state levels of nCLU wild-type, LR, and L343P
mutant proteins are equal when expressed in the Y190 yeasts. The
expression and stability of full-length mouse nCLU wild-type and
nCLU-LR mutant (top panel), as well as nCLU fragment aa
256-448, NcoLR, and L343P mutants of the same fragment
(lower panel) in Y190 cells were assessed by Western blot
analyses using anti-GAL4BD antibody. Expression of proteins in all
experiments described in B were confirmed by Western
blotting.
-helical secondary structure. Thus, human
nCLU appears to have a more tightly compact Ku70 interaction domain
than the comparable domain present in the mouse protein.
-helical structure of the C-terminal
coiled-coil domain in Ku70 binding, we introduced a single proline
residue at position 343 (L343P mutant) in the context of the nCLU aa
256-448 C-terminal half of the protein. When tested by yeast
two-hybrid analyses, this mutant did not show detectable binding to
Ku70. Equal expression of wild-type and L343P mutant proteins in yeast
was confirmed by Western blot analyses (Fig. 4E).
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Fig. 5.
A, relative amounts of
agarose-immobilized proteins used in the GST-pull-down assays. To
estimate the amount of GST and GST-Ku70 proteins used in the in
vitro nCLU-binding assay, glutathione-agarose beads with
immobilized proteins (20 µl of 50% suspension) were heated in the
presence of equal amounts of 2× Laemmli buffer and resolved on the
10% acrylamide SDS-PAGE followed by Coomassie Blue staining.
Lane 1, GST-Ku70 agarose beads; lane 2, GST alone
agarose beads. Bovine serum albumin (BSA) was used as a
blocking reagent and was present in both samples. B, nCLU
binds GST-Ku70 in vitro. Autoradiograph of in
vitro binding of mouse nCLU (aa 18-448) to GST-fused full-length
mouse Ku70 protein and to GST as analyzed by 10% SDS-PAGE as described
under "Experimental Procedures." The lanes from
left to right are as follows: 1, input
protein, 5 µl of the nCLU in vitro translation reaction
mixture; 2, in vitro translated nCLU (from 45 µl of translation mixture) eluted from GST-Ku70-agarose beads,
incubation temperature 23 °C; 3, same as in lane
2, with an incubation temperature of 37 °C; 4,
in vitro translated 35S-nCLU (from 45 µl of
translation mixture) eluted from the GST-agarose beads, incubation
temperature 23 °C; 5, same as in lane 4, with
an incubation temperature of 37 °C; 6, eluted from
GST-Ku70-agarose beads, with an incubation temperature of 4 °C;
7, eluted from GST-agarose beads, with an incubation
temperature of 4 °C. C, leucine residues within the
C-terminal coiled-coil domain of nCLU are necessary for GST-Ku70
binding in vitro. Autoradiograph of the SDS-PAGE (15%
acrylamide) representing in vitro binding of mouse nCLU aa
345-380 to GST-fused full-length mouse Ku70 protein. To confirm the
results of yeast two-hybrid analyses of Ku70 binding to various nCLU
point mutants, the same mutants of mouse nCLU aa 345-380 fragment were
translated in vitro and tested for their ability to interact
with GST-Ku70. Top panel shows the relative input amounts of
in vitro translated wild-type and point mutants of the mouse
nCLU fragment (5 µl of each reaction mixture). Bottom
panel shows GST-Ku70 binding to the respective mouse nCLU protein
fragments retained on the GST-Ku70-agarose beads. 45 µl of each
reaction mixture were used for the binding assay as described
previously (32). The lanes from left to
right are as follows: 1, wild-type nCLU aa
345-380 fragment; 2, LL357,8RQ mutant; 3, E359K
mutant; 4, L361R mutant; 5, D363H mutant; and
6, L371R mutant.
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Fig. 6.
N- and C-terminal coiled-coil domains bind to
each other in yeast two-hybrid system. The human nCLU deletion
mutant containing aa 329-449 (A) or the mouse nCLU deletion
mutant containing aa 18-97 (B) coiled-coil domains were
used as bait to analyze their abilities to bind to various deletion
mutants of mouse nCLU. Numbers represent corresponding amino
acid residues. The potential coiled-coil regions are illustrated as
shaded boxes. Relative strength of protein-protein binding
(plus signs) was indicated as described in Fig.
3A, and all experiments were performed three or more
times.
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Fig. 7.
Subcellular localization of various
GFP-fused nCLU fragments in MCF-7 cells. MCF-7:WS8 cells were
transfected with various GFP-fused mouse nCLU fragments, and
transfectants were analyzed 48 h post-transfection using confocal
microscopy for protein localization and cell death responses. Images in
each row display the following: left (GFP),
GFP-fused nCLU protein; middle (PI), propidium
iodide (PI)-stained DNA; right
(Merged), digitally merged green and
red images; therefore, yellow demonstrates
co-localization. Rows are defined as follows: A,
GFP alone; B and C, GFP-nCLU; D,
GFP-Nterm; E, GFP-Center; F, GFP-Ccoil;
G, GFP-End; and H, GFP-nCLU L343P mutant. Note
the diffuse expression of GFP-nCLU fused proteins in transfections
A, B, D, F, and
G. Note the punctated appearance of GFP-nCLU (C),
GFP-Center (E), and GFP-nCLU L343P mutant (H)
following transfection in MCF-7 cells, and the appearance of two
subpopulation with diffuse or aggregated protein expression following
GFP-nCLU transfection in MCF-7 cells. Also note nuclear localization of
GFP-Nterm and GFP-End. See Table I for quantitation of apoptotic
responses.
Induction of apoptosis by exogenously expressed GFP-fused nCLU protein
fragments
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Fig. 8.
nCLU has two functional NLS motifs.
MCF-7 cells were transfected with GFP-fused mouse Nterm or End
fragments, with or without mutations in the N-terminal and C-terminal
NLSs, respectively. Transfectants were examined 48 h
post-transfection using confocal microscopy. Images in each row display
the following: left (GFP), GFP-fused nCLU
protein; middle (PI), propidium iodide
(PI)-stained DNA; right (Merged),
digitally merged green and red images; therefore,
yellow demonstrates co-localization. Rows are defined as
follows: A, GFP alone; B, GFP-Nterm wild type;
C, GFP-Nterm N-terminal NLS mutant; D, GFP-End
wild type; E, GFP-End C-terminal NLS mutant. See Table II
for quantitation of the percentage of the cells with nuclear
versus cytoplasmic localizations of GFP-fused
proteins.
Mutations in NLS1 or NLS2 result in cytoplasmic localization of
GFP-nCLU constructs
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-site, a site cleaved during maturation of the
60-kDa sCLU precursor protein. nCLU was induced by relatively high
levels of cytotoxic stress (e.g. IR) in direct proportion to
lethality, compared with sCLU protein increases that were very
sensitive to low, nontoxic, and growth stimulatory doses of IR (13).
Utilization of a second downstream AUG start site was proposed to
result in the synthesis of an ~49-kDa precursor nCLU protein that
resided in the cytoplasm of undamaged cells by confocal microscopy
(13). nCLU greatly accumulated and translocated to the nuclei of cells
48-72 h after IR exposure. Overexpression of nCLU, even without IR
treatment, caused cell death (i.e. apoptosis). The mechanism
of synthesis of this nuclear protein from the second AUG translation
start site, and how translation could bypass the much stronger first
translation CCAUG start site present in full-length CLU mRNA, was
not elucidated. Previous investigations did not address how the
precursor ~49-kDa nCLU protein (pnCLU) might reside in the cytoplasm
of undamaged cells in an apparent nonfunctional form (13), and then
dramatically accumulate in the nuclei of damaged cells as an ~55-kDa
mature nCLU death protein 48-72 h after exposure (13). Finally, no previous study attempted to elucidate the functional domains of the
nCLU protein and, in particular, to identify the portions of the
protein required for cell death, Ku70 binding, and/or nuclear localization. In fact, the need for nuclear localization in the cell
death function of the protein was not established.
/
cells, observed with SV40-transformed
murine embryonic fibroblasts (44). The generation of mutant nCLU
proteins with alterations in the C-terminal coiled-coil domain or NLS
should allow us to elucidate the role of nCLU-Ku70 binding in the cell
death functions of nCLU in the future.
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Fig. 9.
A model for nCLU synthesis in human
epithelial cancer cells. CLU promoter activity is stress-inducible
(T. L. Criswell, D. A. Boothman, K. S. Leskov, and D. Y. Klokov,
unpublished data). The precursor transcript of the CLU mRNA is
spliced in various ways, producing the highly abundant sCLU mRNA
(with exon II) and a much lower abundance pnCLU mRNA (without exon
II). sCLU protein is targeted to the ER and secreted outside the cell.
In contrast, the ~49-kDa pnCLU protein stays in the cytoplasm and can
be modified following stress to produce mature 55-kDa nCLU protein when
the radiation dose is equal or exceeds ~1 Gy (42). nCLU is imported
into the nucleus where it binds Ku70 via its C-terminal coiled-coil
domain. Overexpression of nCLU results in caspase-3-independent
apoptosis (13). The C-terminal, Ku70-binding coiled-coil domain is
required for the apoptotic function of nCLU.
Synthesis of nCLU-- Our data show that a low abundance nCLU mRNA is constitutively made in MCF-7 cells, in direct proportion (but in low abundance) to total CLU mRNA. Translation from this nCLU mRNA produces an ~49-kDa nCLU precursor protein (Fig. 9, pnCLU). Detection of this truncated nCLU mRNA in human cells argues against the more unlikely production of the ~49-kDa pnCLU through internal ribosome entry site-mediated translational initiation from a second in-frame AUG sequence in full-length CLU mRNA as suggested (13, 22). There is precedence for alterations in the subcellular localization of protein species via alternative splicing, as proposed for nCLU (Fig. 9). Expression of the rat renin protein undergoes alternative splicing, producing either secreted or intracellular forms of the protein from two unique mRNAs (42). We are investigating the mechanisms of alternative splicing of the CLU mRNA. In particular, we are examining the possible inducibility of nCLU-specific splicing events in response to IR or other cellular stresses. Previous data showed that mature ~55-kDa nCLU protein could be modestly induced (~2-fold) following 1 Gy of IR, compared with more dramatic >50-fold increases in sCLU protein forms. We propose that this reflects the relative abundance of the two mRNA species. Because the truncated, alternatively spliced form of nCLU mRNA was apparent in undamaged cells, our data suggest that the ~49-kDa precursor nCLU protein is constitutively synthesized in cells and stored in a reservoir. The stored protein can then be activated by an as yet unidentified post-translational modification after certain threshold doses of IR (>1 Gy), resulting in an ~55-kDa mature nCLU protein (Fig. 9). Such a mechanism would explain expression of the protein without triggering cell death responses. Localization of pnCLU in the cytoplasm of non-treated cells by confocal microscopy, with no detectable levels in the nuclei of untreated MCF-7 cells (13), appears to support a possible mechanism of sequestration of the pnCLU protein to prevent cytotoxicity.
Functional Domains of the nCLU Protein--
Our data strongly
suggest that nCLU-Ku70 binding was facilitated by strong leucine
zipper-like hydrophobic interactions, mediated by the C-terminal
coiled-coil domain in nCLU and a comparable coiled-coil domain in Ku70.
Deletion of more than one hydrophobic residue in the full-length
C-terminal coiled-coil domain of nCLU was not sufficient to abrogate
nCLU-Ku70 binding (Fig. 3A, deletion mutants
256-344 and 345-380), apparently due to
redundancy of hydrophobic residues required for association with this
protein. Introduction of a single proline residue that was inconsistent with the -helical coiled-coil domain (L343P mutation) completely abrogated Ku70 binding (Fig. 4D) and cell death (Fig.
7H). Thus, the coiled-coil domain is absolutely required for
interaction with Ku70 and initiation of cell death. It is important to
note that the overall length of the C-terminal coiled-coil domain of nCLU is almost twice as large as the dimerization domains of GCN4, c-Fos, c-Jun, and BZLF1. This could explain our finding that deletion of a significant portion of the C-terminal coiled-coil domain of nCLU
did not affect protein-protein interaction.
Our previous studies showed that in-frame deletion of aa residues
492-583 of Ku70 abrogated nCLU-Ku70 binding. Recent structural data on
the Ku complex (43) indicated that this deletion affected -helices
15, 16, and 16' that also contained 4-3 patterns of hydrophobic amino
acids (Fig. 4A). The ultimate role of the 4-3 repeated
hydrophobic residues in mediating Ku70 binding was elucidated using
point mutation analyses of the 345-380 minimal Ku70-interacting fragment of nCLU. Thus, the interaction between Ku70 and nCLU appears
to occur through the C-terminal coiled-coil domain of nCLU, a region of
the protein also required for cell death (only in conjunction with the
C-terminal NLS of nCLU) in transfected cells.
At the present time, little is known about the regulation of nCLU
induction and translocation. Translocation of pnCLU from the cytoplasm
to the nucleus was accompanied by an electrophoretic mobility shift of
the nCLU protein (13). In its inactive form, pnCLU was an ~49-kDa
protein present in the cytoplasm of control, non-irradiated MCF-7
cells; little, if any, protein was found in the nuclei of control MCF-7
cells (13). After IR exposure, ~55-kDa nCLU protein levels increased
and translocated from the cytoplasm into the nuclei of damaged cells
(13). The IR-induced, post-translational modification processes that
activate nCLU are not known, but we hypothesize that these
modifications are involved in exposing either of the functional NLS
signals, allowing subsequent translocation of pnCLU to the nucleus, as
the mature nCLU death protein (Fig. 9). It is also possible that other
proteins may be involved as specific inhibitors of pnCLU maturation in
much the same way as IB blocks NF-
B activation (44). The nature of the apparent post-translational modification of pnCLU is not known,
but mass spectrometry studies are ongoing to elucidate these
modification processes.
Comparison of the subcellular distribution of GFP-nCLU and GFP-Center suggests that cytosolic pnCLU may be actively prevented from entering the nucleus or actively excluded. The non-coiled central region of the pnCLU protein (amino acids 98-317) appears to be responsible for the inhibition of nuclear import and the formation of the characteristic aggregates of nCLU in the cytoplasm; this region of the protein may bind to a certain cytosolic factor(s), may be required for oligomerization, or needed for folding of the pnCLU protein in the cytosol. The distinct nuclear localization of the End fragment of nCLU (aa 368-448) and significantly lower nuclear expression of the GFP-End NLS2mut (Fig. 7G and Fig. 8, D and E; Table II) indicate that the C-terminal NLS was the functional nuclear import sequence for nCLU. N-terminal NLS1 may contribute to the nuclear translocation of nCLU, but our data on Fig. 8, B and C, and Table II demonstrate that NLS1 function is not as prominent as that of NLS2.
Because the C-terminal coiled-coil domain of mouse nCLU (aa 318-367) did not have an apparent NLS, very little of the protein was detected in the nucleus of cells with apoptotic morphology. These data indicate that enough of this protein may have entered the nucleus due to forced, cytomegalovirus-directed overexpression to cause cell death. An alternative hypothesis is that the cell death function of nCLU may not require nuclear translocation. Our data suggest that dissociation of pnCLU coiled-coil domains may allow modification, translocation, and cell death responses. We are currently exploring the mechanism of nCLU transport into the nucleus, as well as the role of its nuclear internalization in nCLU-mediated cell death responses, including the apparent triggering of apoptotic responses.
Function of nCLU as a Death Gene--
The precise intracellular
functions of nCLU, as well as the significance of its binding to Ku70
protein, remain to be determined. As demonstrated in our previous (13)
and current studies, overexpression of GFP-nCLU induces apoptosis in an
apparent C-terminal coiled-coil- and NLS-dependent fashion.
For example, overexpression of GFP-Ccoil, the Ku70-binding fragment of
nCLU, resulted in short term apoptotic cytotoxicity (Fig. 7 and Table
I), as well as long term clonogenic cytotoxicity (13). Although overall
apoptosis measurements did not exceed 25-30% at any time, the
clonogenic survival of the GFP-nCLU-positive MCF-7 cells was ~10-fold
lower than GFP-alone transfected MCF-7 cells. Our data suggest, but do
not prove, that interaction and interference with
Ku70-dependent end joining, even in the absence of overt IR
damage, can cause significant cell death. Superficially, this appears
to mimic the enhanced apoptosis and lowered plating efficiency of
Ku70/
mouse embryonic fibroblasts (44).
Elucidation of mechanisms by which CLU gene
expression and nCLU production occur after DNA damage in tumor cells
may augment various cancer therapies.
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ACKNOWLEDGEMENTS |
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We thank Dr. John J. Pink for stimulating discussions and helpful suggestions, Dr. Martin Tenniswood for mouse CLU cDNA, Dr. M. Abe for mouse Ku70 cDNA, and Dr. Shinako Araki for help with confocal microscopy. We are also grateful to the Case Western Reserve University-University Hospital Comprehensive Cancer Center Radiation, Flow Cytometry, DNA Sequencing, and Confocal Core Facilities supported by NCI Grant P30 CA43703 from the National Institutes of Health.
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FOOTNOTES |
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* This work was supported by NCI Grants CA78530 (to D. A. B.) and CA84578 (to T. J. K.) from the National Institutes of Health and by the Department of Defense Breast Cancer Research Program DAMD17-01-1-0196 (to K. S. L.).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.
To whom correspondence should be addressed: Dept. of Radiation
Oncology (BRB-326 East), Laboratory of Molecular Stress Responses, Case
Western Reserve University, 10900 Euclid Ave., Cleveland, OH
44106-4942. Tel.: 216-368-0840; Fax: 216-368-1142; E-mail: dab30@po.cwru.edu.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M209233200
2 C.-R. Yang, K. S. Leskov, and D. A. Boothman, unpublished observations.
3 K. S. Leskov and D. A. Boothman, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
CLU, clusterin;
aa, amino acid;
nCLU, nuclear clusterin;
sCLU, secreted clusterin;
pnCLU, precursor of nuclear clusterin;
GFP, green fluorescent protein;
GST, glutathione sulfotransferase;
MCF-7, MCF-7:WS8;
IR, ionizing radiation;
ER, endoplasmic reticulum;
N-term, N-terminal domain of nCLU (aa
18-97);
Center, central region of nCLU (aa 98-317);
Ccoil, C-terminal
coiled-coil region of nCLU (aa 318-368);
End, C-terminal non-coiled
coil region of nCLU (aa 367-448);
NLS, nuclear localization signal;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
RT, reverse transcriptase;
ER, endoplasmic reticulum;
Gy, gray.
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