From the Laboratory of Molecular Clinical Chemistry,
Institute for Chemical Research, Kyoto University, Gokasho, Uji,
Kyoto 611-0011, Japan, the § Department of Hygiene, Kyoto
Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan, ¶ Department of Surgery and Surgical Basic Science,
Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto
606-8507, Japan, and the
Department of Anatomy Kyoto Prefectural
University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan
Received for publication, November 10, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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We isolated cDNAs encoding a novel RING
finger protein (LUN), the mRNAs of which were expressed at high
levels in the lung. In situ hybridization revealed that LUN
mRNAs were expressed in the alveolar epithelium of the lung. The
LUN gene locus was assigned to chromosome 9p21, which
contains candidate tumor suppressor genes associated with loss of
heterozygosity in more than 86% of small cell lung cancers. We
clarified that LUN is localized to the nucleus and reveals
Zn2+-dependent DNA binding activity. The region
from amino acids 51 to 374 of LUN is responsible for DNA binding.
Furthermore, we identified a novel palindromic binding consensus
(5'-TCCCAGCACTTTGGGA-3') for the LUN binding.
Interestingly, this LUN binding palindromic sequence is found in the
upstream transcriptional regulatory region of the E-cadherin gene and
two intervening regions of the talin gene. Our results
suggested that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes.
The RING1 finger motif
is one example of a Zn2+ binding domain that is found in
proteins from viruses to vertebrates and defines a superfamily of
diverse proteins (1, 2). The motif itself is distinct from classic zinc
finger motifs in terms of sequence homology, Zn2+ binding
scheme, and three-dimensional structure (3-5). The RING finger motif
appears to mediate both protein-DNA (6-8) and protein-protein interactions (2) and, in some cases, E2-dependent
ubiquitination (9, 10). The RING finger proteins are thus involved in a variety of fundamental cellular roles such as gene regulation, oncogenesis, viral pathogenicity, embryogenesis, V(D/J)
recombination, DNA repair, and signal transduction (1, 2).
There are a number of RING finger proteins with oncogenic potential,
including the protein encoded by the breast cancer susceptibility gene
BRCA1 (11), an acute promyelocytic leukemia-associated protein (PML) (12, 13), a RET finger protein (RFP) (14), c-Cbl and
Bmi-1 proto-oncoproteins (15-17), and Mel-18, a nuclear DNA-binding
protein isolated from melanomas (6). Among them, Mel-18 and Bmi-1 are
highly homologous human oncogene products (18). Mel-18 possesses a
tumor suppressor activity by acting as a negative regulator of
transcription and is found in all tumor cells at increased levels (6,
7). Bmi-1 is morphogenic during embryonic development and hematopoiesis
and cooperates with c-Myc in oncogenesis (16, 17). PML and RET finger
protein (RFP) become oncogenic when found as fusion proteins resulting
from chromosomal translocations (12-14). PML is found in a form fused to the retinoic acid receptor In industrialized countries, lung cancer is a leading cause of cancer
death. One of our research interests is to identify important RING
finger family genes including oncogenes and tumor suppressor genes in
lung alveolar cells. In this study, we employed the PCR approach and
degenerate primers corresponding to the RING finger motif. Here, we
describe the isolation and characterization of LUN, a new member of the
RING finger proteins family that might be associated with tumorigenesis
and/or tumor suppression. LUN mRNAs are expressed at high levels in
alveolar epithelium of the lung. The LUN gene is localized
to chromosome 9p21, which contains candidate tumor suppressor genes
associated with loss of heterozygosity in more than 86% of small cell
lung cancer. LUN is a 119-kDa nuclear protein and shows
Zn2+-dependent and sequence-specific DNA
binding ability. A novel palindromic binding consensus sequence for
LUN51-374 was found in the upstream transcriptional
regulatory region of the E-cadherin gene and introns of the
talin gene. Our findings provide an important clue in the
further study of the function of LUN and the mechanism of its
involvement in tumorigenesis in small cell lung cancer (SCLC).
Cloning of the LUN cDNA--
First-strand cDNA was
prepared from human lung poly(A)+ RNA
(CLONTECH). This cDNA was then used as a
template for PCR with an oligo(dT) primer and two degenerate primers
(5'-TG(T/C) CTI CA(T/C) TCI TT(T/C) TGC-3' and 5'-TG(T/C) TT(A/G)
CA(T/C) AG(T/C) TT(T/C) TGC-3') homologous to an amino acid sequence
(CLHSFC) that is conserved among several members of the RING finger
family. The PCR products were hybridized with a second degenerate
oligonucleotide (5'-CA(T/C) TCI TT(T/C) TGI AA(A/G) TCI TGC-3')
homologous to an overlapping region of the RING finger domain
(HSFCKSC). DNA from random positive clones was sequenced and compared
with the GenBankTM data base. Two of the clones contained
partial cDNAs for a novel RING finger protein, which we named LUN.
The full-length human LUN cDNA was generated by 5'-rapid
amplification of cDNA ends using a Marathon cDNA amplification
kit (CLONTECH). The cDNA was reverse-transcribed from poly(A)+ mRNA from human lung,
brain, the glioma cell line T98 or T-cell line Molt-4 using the
cDNA synthesis primer provided with the kit. The first round of PCR
was carried out using an adapter primer 1 and gene-specific reverse
primer (5'-TAACTCGAGCACCAGCACGATAAAG-3'). The second round of PCR was
performed using the first round gene-specific PCR amplification product
as the template, adapter primer 2, and a nested gene-specific primer
(5'-CAAAGATCTTTCATCTGCCGTAGTTG-3'). The cDNA amplified by 5'-rapid
amplification of cDNA ends was then subcloned and sequenced.
Fluorescence in Situ Hybridization (FISH)--
Replication
G-banded chromosomes were prepared from the
phytohemagglutinin-stimulated lymphocytes from a normal male donor using the thymidine synchronization/bromodeoxyuridine release technique (20). Just before hybridization, the chromosome slides were
denatured with 70% formamide in 2× sodium saline citrate at 70 °C
for 2 min. The 3549-bp fragment (corresponding to nt 270-3818 of the
LUN cDNA) was labeled with biotin-16-dUTP (Roche Molecular
Biochemicals) by nick translation. The probes were denatured at
75 °C for 10 min and then mixed with an equal volume of
hybridization buffer (20% dextran sulfate in 4× sodium saline
citrate). The hybridization mixture was placed on the denatured
chromosome slides. After overnight hybridization, signal detection was
achieved in three amplification steps, one with avidin-fluorescein
isothiocyanate (Vector), one with biotinylated anti-avidin D (Vector),
and one with avidin-fluorescein isothiocyanate. Chromosomes were
counter-stained with propidium iodide. FISH signals and the replicate
G-bands for the same metaphase were detected under fluorescence
microscopy (Nikon) through a B-2A filter and a UV-2A filter,
respectively, and photographed separately. The precise assignment of
the LUN gene to chromosomal bands was achieved by
superimposing FISH signals over the G-banded chromosomes (21).
Northern Blotting Analysis--
Northern blots containing 2 µg
of poly(A)+ RNA from several different adult human tissues
(Invitrogen) were probed for expression of LUN mRNA as previously
described (22). The probe, a 3549-bp fragment (corresponding to nt
270-3818 of the LUN cDNA), was random-primed with
[ In Situ Hybridization--
To generate probes for in
situ hybridization, we cloned a 236-bp BamHI fragment
(nt 1-236) and a 588-bp BamHI fragment (nt 270-857) of the
LUN cDNA into the pBluescript II KS( DNA Transfection--
The BamHI to EcoRI
fragment of the cDNA encoding LUN51-1045 was ligated
into the BglII and EcoRI sites of the mammalian expression vector pEGFP-N1 (CLONTECH), which allows
in-frame fusion with enhanced Aequorea victoria green
fluorescent protein (GFP) under the control of cytomegalovirus
immediate early promoter and contains SV40 polyadenylation signals
(referred to as pGFP-LUN51-1045). HeLa cells were seeded
on chamber slides and incubated for 16 h. Cells were then washed
with Dulbecco's modified Eagle's medium twice and transfected with
1.4 µg of plasmid DNA by LipofectAMINE (Life Technologies)-mediated
transfection in Dulbecco's modified Eagle's medium. A 3-fold volume
of 15% fetal bovine serum in Dulbecco's modified Eagle's medium was
added 3 h later. The transfection mixture was removed 21 h
later, and the cells were maintained in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum for an additional 24 h.
At 48 h after transfection, confocal imaging analysis was
performed using a Zeiss LSM510 laser-scanning microscope. For GFP
visualization, an fluorescein isothiocyanate filter set was used.
Expression and Purification of Glutathione S-Transferase (GST)
Fusion Proteins--
Segments of the LUN cDNA encoding aa
51-1045, and aa 51-374 were cloned into the BamHI and
EcoRI/SmaI sites of the vector pGEX-2T (Amersham
Pharmacia Biotech) to generate the plasmids pGST-LUN51-1045 and pGST-LUN51-374,
respectively. Each plasmid produces an in-frame fusion of GST with
LUN51-1045 or LUN51-374 (containing RING
finger and leucine zipper coiled-coil domains). For isolation of GST
fusion proteins, transformed bacteria were grown to early log phase and
induced for 3 h with 0.1 mM
isopropyl-1-thio- DNA Binding Assay--
Each 125I-labeled GST fusion
protein (6 × 105 cpm, 2 pmol) was diluted in 600 µl
of binding buffer A (10 mM HEPES-NaOH, 50 mM KCl, 1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1) and
then incubated with 15 µl of calf thymus double-stranded
DNA-cellulose or single-stranded DNA-cellulose (Sigma) in the presence
of 1 mM ZnCl2, 1 mM
MgCl2, 1 mM CaCl2, 1 mM
MnCl2, and/or 1 mM EDTA alone or in combination at 4 °C for 1 h with gentle shaking. After centrifugation, the pellets were washed three times with the same binding buffer A. The
remaining radioactivity on the DNA-cellulose was measured by
Construction of Genomic Library and Screening for LUN Binding
Sequences--
Human genomic DNA was digested to completion with
MboI. The resultant mixture of digestion products was
ligated into the BamHI site of the plasmid pUC118 followed
by electroporation into competent DH5 Gel Mobility Shift Assay--
For preparation of a DNA probe,
the synthetic oligonucleotide 5'-CCTGTAATCCCAGCACTTTGGGAGGCTGAGG-3' and
its complementary oligonucleotide were end-labeled with T4
polynucleotide kinase in the presence of [ Cloning of LUN cDNAs--
We sought to identify novel proteins
of the RING finger family expressed in the lung. We used a PCR strategy
with degenerate oligonucleotides to isolate partial cDNA clones of
RING finger proteins using human lung cDNA as a template. Two of
the partial cDNA clones isolated in this fashion encoded a novel
RING finger protein, which we designated as LUN based on its unique
expression pattern (see below). The cDNA clones isolated by
PCR were subsequently used to generate 5'-overlapping clones by
5'-rapid amplification of cDNAs ends. Fig.
1A shows the complete
nucleotide sequence and the predicted amino acid sequence of LUN
cDNA. The LUN cDNA from lung and brain consisted of 3818 nt and
contained a long open reading frame with one potential translational
initiation codon (ATG) and in-frame upstream stop codons at the 5' end
of the cDNA (GenBankTM accession number AB045732). The
sequence surrounding this ATG codon is in a favorable context for
translational initiation as defined by Kozak (24). We also isolated a
3623-bp LUN cDNA with deletion of nt 123-317, possibly due to
alternative splicing from the lung, the glioma cell line T98, and
T-cell line Molt-4 (GenBankTM accession number
AB045733).
During the course of this study, 5055-bp and 3264-bp human cDNA
clones that matched our cDNA clones were reported by Zhou et
al. (25) and Haluska et al. (26), respectively.
However, the 5055-bp human cDNA isolated as a p53BP3
(p53-binding protein 3) cDNA (25) was
inconsistent with our clones in the 5' end 1247 nt, which completely
matched different human cDNA clones, NH0469M07 and
RP11-422L5 (GenBankTM AC005037 and AC037455,
respectively). On the other hand, although the 3264-bp human cDNA
isolated as a Topors (topoisomerase I-binding
RS protein) cDNA (GenBankTM AF098300) (26)
lacked a 3'-untranslated region of 555 nt and polyadenylation signal,
it matched our complete cDNA sequence (3818 and 3624 bp) almost
exactly, except for a short deletion due to alternative splicing (Fig.
1A).
The predicted LUN protein consists of 1045 aa, with a calculated
molecular mass of 119 kDa and pI 9.8. Sequence analysis revealed several distinctive features (Fig. 1, A and B).
The most striking sequence of LUN is the RING finger motif (aa
103-141). The RING finger is one example of a Zn2+ binding
motif defined by a conserved pattern of cysteine and histidine residues
(C3HC4) that is found in a wide variety of proteins of diverse origins
and functions (Fig. 1C). The region from aa 293-365 is
likely to adopt a leucine zipper and a coiled-coil structure. The
N-terminal region (aa 51-374) containing a RING finger motif, a
putative leucine zipper, and a predicted coiled-coil structure revealed
a Zn2+-dependent and sequence-specific DNA
binding activity (see below). There are five PEST sequences at
aa 13-28, 386-432, 490-509, 921-950, and 999-1024. Two putative
bipartite nuclear localization signals are present near the middle of
the protein, indicating that LUN may function in the nucleus. In
addition, a region rich in serine and arginine residues and two
putative small ubiquitin-like protein (SUMO-1) modification
sites ( Chromosomal Localization of the Human LUN Gene--
To confirm the
chromosomal mapping of the LUN gene, we performed FISH
analysis on metaphase chromosomes from normal male lymphocytes using
the 3549-bp LUN cDNA probe (corresponding to nt 270-3818). Several
independent hybridizations exhibited a specific twin-spot signal on the
short arm of a medium-sized chromosome, consistent with chromosome 9 on
the basis of G-banding pattern (Fig. 2,
A and B). No twin-spot signals were observed on
other chromosomes. Fine analysis of 8 specifically hybridized
chromosomes mapped the LUN gene to the p21 region of
chromosome 9 (Fig. 2, C-E).
Expression of LUN mRNAs in Human Tissues--
As described in
the cloning of LUN cDNAs (Fig. 1A), we cloned the 3.8-kb
LUN cDNA from adult human lung and brain and the 3.6-kb cDNA
from lung, T98 cell line, and Molt-4 cell line. Northern blotting
analysis was performed to examine the expression of LUN mRNAs in
adult human tissues (Fig. 3). Two LUN
mRNAs were detected differentially among the tissues: a 3.8-kb
mRNA in the brain, kidney, liver, lung, spleen, pancreas, and
skeletal muscle and a 3.6-kb mRNA in the heart and lung. Human LUN
mRNAs were most highly expressed in the lung. Moderate expression
with variation was detected in the heart, brain, kidney, liver, spleen,
and skeletal muscle. Little expression of LUN mRNA was detected in
the pancreas. Thus, it was remarkable that the highest level of LUN
expression occurred in the lung.
Cellular Localization of LUN mRNA in the Lung--
As shown in
Fig. 3, LUN mRNAs were expressed at much higher levels in the lung
than in other tissues. To better localize the lung cells expressing LUN
mRNA, we performed in situ hybridization with adult lung
sections (Fig. 4). Hybridization with the
antisense LUN probe revealed specific signals in the alveolar
epithelium (Fig. 4A), whereas the sense probe did not show
any signals in the lung tissue tested (Fig. 4C). In
particular, not all, but a number of squamous cells (type I) and
cuboidal cells (type II), revealed specific signals of LUN mRNA in
the cytoplasm (Fig. 4B).
Subcellular Localization of LUN Protein--
Because the LUN
protein contains two putative bipartite nuclear localization signals
(aa 616-645), it might function in the nucleus. To investigate the
subcellular localization of LUN, HeLa cells were transformed with a
plasmid expressing GFP alone or GFP-LUN fusion protein by DNA
transfection. As shown in Fig. 5, cells
expressing GFP-LUN exhibited nonhomogeneous fluorescent signals in the
nucleus (panels A, C, and E). The
nuclear localization pattern of LUN was similar to those of RING finger
proteins, Mel-18, PML, RPT-1, and Vmw110 (6, 29-31). In contrast, the
cells expressing GFP alone displayed diffuse fluorescence in both the
cytoplasm and the nucleus (Fig. 5, panels B,
D, and F). These results clearly indicated that
LUN was localized to the nucleus.
DNA Binding Property of the LUN Protein--
More than 80 RING
finger proteins have been identified to date; however, the cellular
functions of the RING finger are not well understood (2). Evidence has
accumulated in recent years indicating that this motif
is structurally diverse and is involved in protein-DNA interaction
(6-8), protein-protein interaction (2-4), and
E2-dependent ubiquitination (9, 10). To explore the ability
of DNA binding of LUN by its RING finger, we prepared two kinds of
recombinant LUN products, GST-LUN51-1045, which covers
almost the full-length of the molecule, and
GST- LUN51-374, which contains the RING finger and
a leucine zipper and coiled-coil region (Fig.
6). After 125I-labeling, we
examined the binding ability of GST-LUN51-1045 and
GST-LUN51-374 to double-stranded or single-stranded DNA-cellulose in the presence or absence of mixed divalent cations (1 mM each of Zn2+, Mg2+,
Mn2+, and Ca2+). As shown in Fig.
7A, both
GST-LUN51-1045 and GST-LUN51-374 exhibited an
~10-fold higher level of binding to double-stranded DNA-cellulose in
comparison with GST in the presence of mixed divalent cations, whereas
little or no background binding was observed in the presence of
1 mM EDTA. Similar binding of GST-LUN51-1045 and GST-LUN51-374 to single-stranded DNA-cellulose,
depending on divalent cations, was also observed (Fig. 7B).
Thus, LUN, especially LUN51-374 containing the RING finger
motif and a leucine zipper and coiled-coil region, was capable of
binding to DNA.
Because (i) divalent cations were required for DNA binding by LUN (Fig.
7) and (ii) Zn2+ is known to be necessary for autonomous
folding of the RING finger motif (5), we investigated the effects of
divalent cations on DNA binding of LUN in detail. As shown in Fig.
8A, the presence of a single
metal ion (1 mM each) revealed background levels of double-stranded DNA binding of LUN, whereas double-stranded DNA binding
was completely restored by the addition of 1 mM
Zn2+ in combination with one of three other metal ions.
Although the combinations of two of Mg2+, Mn2+,
and Ca2+ exhibited low levels of DNA binding, these
combinations without Zn2+ could not completely cover the
DNA binding ability of LUN. Similar results were also obtained in
single-stranded DNA binding assays (Fig. 8B). These results
indicated that Zn2+ with either Mg2+,
Mn2+, or Ca2+ is essential for the
binding of LUN to DNA, probably by maintaining the structural integrity
of the RING finger motif.
Determination of LUN Binding Sequences from Human Genomic
DNA--
To isolate human DNA elements capable of sequence-specific
interaction with LUN, a library of MboI-digested small
genomic DNA fragments was subjected to three consecutive cycles of
in vitro binding selection with glutathione-Sepharose beads,
which retained the GST-LUN51-1045 and
GST-LUN51-374 fusion proteins. After the third selection
cycle, 72 independent clones were isolated, and the inserted genomic
fragments were sequenced. The average insert was 548 bp in length
(range 47-1380 bp). Computer analysis of the sequences of the 72 clones revealed that 55 clones showed significant homologies with each
other and were classified into three groups (Fig.
9). Each of the 25 binding clones in
group A contained inverted copies of the 5-bp motif, 5'-TCCCA-3' and was capable of forming a palindromic structure with a 6-bp intervening loop (Fig. 9A). In contrast, each of the binding clones in
groups B (22 clones) and C (8 clones) contained one copy of the 5-bp motif with a few bases of the other half of the palindromic structure (Fig. 9, B and C). These results clearly
demonstrated that LUN is a sequence-specific DNA-binding protein like
Mel-18, a RING finger-containing transcriptional negative regulator
(7), and RAG-1, a RING finger-containing V(D/J) recombination
mediator (32, 33). Furthermore, the consensus LUN binding sequence and
its surrounding sequences encompass the well characterized cis elements, 5'-TTTGGGAG-3' (for Lyf-1 and
Ik-2, mammalian lymphocyte-specific transcriptional regulators; Fig. 9,
A and C) (34, 35),
5'-TCTAATCCC-3' (for Bcd, a
Drosophila transcriptional regulator; Fig. 9, A
and B) (36, 37), 5'-CTGGGAGT-3', and
5'-ACTCCCAG-3' (human erbB-2 promoter; Fig. 9A) (38). However, no homology was found in
the binding sequences of Mel-18 and RAG-1 (7, 32, 33). To our surprise,
the LUN binding palindromic sequence of 33 bp,
5'-GCCTGTAATCCCAGCACTTTGG-GAGGCTGAGGC-3', completely matched the sequence (
For further verification of sequence-specific DNA binding of LUN, we
performed gel mobility shift assay using a series of synthetic
oligonucleotides corresponding to the binding consensus (C1-C5) and
GST-fused recombinant LUN proteins. As shown in Fig. 10, a gel mobility shift assay revealed
a retarded DNA complex with GST-LUN51-1045 or
GST-LUN51-374 (panels B and C,
lane 2), whereas the addition of GST had no effect
(panel A, lane 2). The addition of a 200-fold
excess of unlabeled identical oligonucleotide C1 or C2 abolished the
band shift almost completely (panels B and C,
lane 3 and 4). These results clearly indicated that LUN is capable of binding to the 22-bp palindromic sequences containing two copies of the 5-bp motif. Deletion mutant
oligonucleotides C3 and C4 corresponding to each half-site of the
palindromic structure also significantly competed with C1 binding, but
this effect was not complete (panels B and C,
lanes 5 and 6). Oligonucleotide C5 corresponding
to the 5-bp motif was scarcely capable of competing formation of the
retarded complex (panels B and C, lane
7). Thus, LUN had a much higher affinity to C1 oligonucleotide
than to C5 oligonucleotide. These results, therefore, indicated that
LUN has the propensity to bind to the palindromic structure containing inverted copies of the 5-bp motif, and the surrounding sequences of the
5-bp motif are indispensable for the interaction between LUN and
DNA.
In the present study, we isolated cDNAs encoding a novel RING
finger protein, LUN, the mRNAs of which were expressed at high levels in the alveolar epithelium of the lung. The high level of LUN
expression in the lung indicated that this molecule plays an important
role(s) in the lung. We attempted to characterize the biochemical
properties and functions of LUN. With regard to the cellular functions
of LUN, it is important that several nuclear RING finger proteins,
Mel-18, RAG-1, PML, RPT-1, RAD-18, and RING1, are involved in the
regulation of transcription and chromatin structure (6, 7, 29, 30, 32,
33, 40, 41). The localization of a GFP-LUN fusion protein in
punctate nuclear subdomains is consistent with the predicted nuclear
function(s) of LUN. In fact, we clarified that the N-terminal RING
finger-containing region (aa 51-374) of LUN specifically binds to a
novel palindromic consensus sequence
(5'-TCCCAGCACTTTGGGA-3') and genomic fragments containing the 5-bp core motif (5'-TCCCA-3') in a
Zn2+-dependent manner. Furthermore, the LUN
binding palindromic sequence and its surrounding sequence
(5'-GCCTGTAATCCCAGCACTTTGGGAGGCTGAGGC-3') completely matched the sequence of the upstream transcriptional regulatory region ( In general, nuclear palindrome-binding proteins such as
Maf, Jun, Fos, and PBP form leucine zipper-mediated homodimers
and heterodimers that are important for their functions (38, 47-50). On the basis of the specific DNA binding data presented here, it seems
possible that LUN forms a homodimer for palindromic sequence binding
through its leucine zipper and coiled-coil region. Moreover, it is also
possible that LUN forms several types of heterodimers with distinct DNA
binding specificities because (i) many genomic LUN binding clones
containing the half-site of the palindromic structure with the 5-bp
core sequence were isolated and (ii) a p53 binding and topoisomerase I
binding domain has been identified in the C-terminal half of the
protein (25, 26). Interestingly, LUN contains two putative SUMO-1
modification sites in the p53 binding and topoisomerase I binding
domain, and both p53 and topoisomerase I are modified with SUMO-1
in vivo (27, 51). Because SUMO-1 modification is known to
affect the ability of the modified protein to interact with target
proteins (27, 28, 51), the interaction of LUN with p53 and/or
topoisomerase I, resulting in distinct DNA binding specificities, may
be regulated through SUMO-1 modification.
We show that the human LUN gene was localized to chromosome
9p21. More than 86% of SCLC exhibit loss of heterozygosity at 9p21
(52), suggesting the existence of tumor suppressor genes within the
lost region. Based on mapping of the LUN gene to 9p21 and
its expression at high levels in alveolar epithelium, LUN is
a candidate tumor suppressor gene related to SCLC. SCLC is distinct
from other types of lung cancer accompanied by metastasis. Cancer
metastasis and invasion are closely associated with cell-to-cell and
cell-to-extracellular matrix adhesiveness. Down-regulation of
E-cadherin has been reported to occur in various cancers including SCLC
and other lung cancers (39, 42, 53) and to parallel with tumor
progression toward a malignant invasive state (43, 44). As discussed
above, LUN is capable of binding specifically to the transcriptional
regulatory region of the E-cadherin gene and is expressed at high
levels in the alveolar epithelium. The present results suggest a
possible molecular mechanism of SCLC in which loss of the
LUN gene would cause loss of trans-activation by LUN, resulting in transcriptional inactivation of the
E-cadherin gene.
In this study, we isolated cDNAs encoding a novel RING finger
protein, LUN, the mRNAs of which are expressed at high levels in
alveolar epithelium of the lung. The LUN gene locus was
assigned to the chromosome 9p21, which contains candidate tumor
suppressor genes associated with loss of heterozygosity in more than
86% of SCLC. LUN localizes to the nucleus and binds specifically to a
novel palindromic binding consensus
(5'-TCCCAGCACTTTGGGA-3') in a
Zn2+-dependent manner. The sequence from amino
acids 51-374 of LUN is responsible for palindrome binding. The LUN
binding palindromic sequence was found in the upstream transcriptional
regulatory region of the E-cadherin gene and in two intervening regions
of the talin gene, suggesting that LUN might be an important
trans-acting transcriptional regulator for lung
cancer-associated genes including E-cadherin and talin
genes. The physiological and clinical significance of LUN remains
unclear. However, the findings described in this report will be
important for further intensive studies on transcriptional regulation
of E-cadherin and talin genes, lung development,
differentiation, and tumorigenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in patients with acute promyelocytic leukemia, and RET is comprised of the RET (RFP) finger protein fused to
a tyrosine kinase domain. Human c-Cbl becomes oncogenic after deletion
of the C-terminal region including the RING finger motif, which links
the loss of a functional RING finger with tumorigenesis (15). Two
predisposing mutations in BRCA1 result in deletion of the
RING finger domain and a point mutation in one of the RING finger
Zn2+ ligands (11, 19). Again, this links the loss of a
functional RING finger with tumorigenesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (220 TBq/mmol; Amersham Pharmacia Biotech)
using a Megaprime random primer labeling kit (Amersham Pharmacia
Biotech). Hybridization was performed at 42 °C with the radiolabeled
LUN cDNA probe. After hybridization, the blots were washed at
65 °C for 10 min with 2× sodium saline citrate containing 0.1% SDS
and for 30 min with 0.1× sodium saline citrate containing 0.1% SDS.
The probed blots were subjected to autoradiography with intensifying
screens at
80 °C.
) vector (Stratagene),
respectively. The plasmids were linearized with EcoRI and
transcribed with T7 polymerase to yield an antisense RNA probe or
linearized with NotI and transcribed with T3 polymerase to
yield a sense RNA probe. Transcription reactions included
digoxigenin-UTP (Roche Molecular Biochemicals) as a label. A human
multi-tissue set was purchased from Novagen. Hybridization and high
stringency washes were carried out according to established procedures
(23). After deparaffinization and acetylation, digoxigenin-labeled
probe was hybridized at a final concentration of 0.5 µg/ml. After the final high stringency wash, slides were washed successively in digoxigenin buffer (100 mM Tris-HCl, 150 mM
NaCl, pH 7.5) and digoxigenin buffer containing 1.5% blocking reagent
and reacted with alkaline phosphatase-conjugated anti-digoxigenin
(1:500 dilution; Roche Molecular Biochemicals) in the dark. The slides
were then washed in digoxigenin buffer and alkaline phosphatase buffer
(100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.0). The colorimetric reaction
product was developed at room temperature for 18 h with 50 µg/ml
5-bromo-4-chloro-3-indolyl phosphate and 75 µg/ml nitro blue
tetrazolium in alkaline phosphatase buffer.
-galactoside. The resuspended bacterial pellet was
then sonicated in lysis buffer (25 mM Tris-HCl, 1% Triton
X-100, 1 mg/ml lysozyme, 5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 50 µM
p-amidinophenylmethanesulfonyl fluoride hydrochloride, pH
8.0). The supernatant was then incubated with glutathione-Sepharose 4B
beads (Amersham Pharmacia Biotech) for 12 h at 4 °C and washed
with phosphate-buffered saline three times. Finally, the GST fusion
proteins were eluted with elution buffer (50 mM Tris-HCl,
10 mM glutathione, pH 8.0). The GST fusion proteins were
radiolabeled at their N-terminal amino group and
-amino group of
lysine residues using
N-succinimidyl-3-(4-hydroxy-3,5-di[125I]iodophenyl)
propionate (163 TBq/mmol; PerkinElmer Life Sciences) according
to the procedure recommended by the manufacturer. The specific
activities of 125I-labeled proteins were adjusted to
~3 × 105 cpm/pmol.
-counting.
bacteria, which were then
grown in mass liquid culture. Plasmid DNA was then extracted from the
cultured bacteria and used for screening. The primary library, taken
for the first round of screening, contained 5.9 × 106
clones, 80% of which contained genomic DNA inserts (data not shown).
For screening, 20 µg of plasmid DNA from the library was mixed with
GST-bound glutathione-Sepharose beads in binding buffer B (10 mM HEPES-NaOH, 50 mM KCl, 1 mM
ZnCl2, 1 mM MgCl2, 1 mM
2-mercaptoethanol, 20% glycerol, pH 7.1). After incubation for 1 h at 4 °C, unbound DNA was recovered by centrifugation. Then,
GST-LUN51-1045- or GST-LUN51-374-bound
glutathione-Sepharose beads were added and incubated for 1 h at
4 °C. The beads were then washed five times with washing buffer
(identical to the above binding buffer B except for 2% glycerol). The
DNA was released from the beads by incubation for 10 min at 45 °C in
a solution containing 1% SDS and used to transform DH5
. This
screening was repeated twice to generate a more enriched library. After
the third screening, 72 colonies were picked up, and plasmid DNA was
extracted from each colony. The inserted genomic fragments were
purified and sequenced.
-32P]ATP
(111 TBq/mmol; PerkinElmer Life Sciences), annealed, and purified with
MicroSpin G-25 columns (Amersham Pharmacia Biotech). Each reaction
mixture contained 8 pmol of 32P-labeled probe (~4 × 104 cpm) and 8 pmol of GST fusion protein in binding buffer
C (10 mM HEPES-NaOH, 50 mM KCl, 1 mM ZnCl2, 1 mM MgCl2, 1 mM 2-mercaptoethanol, 20% glycerol, 5 µg/ml aprotinin,
0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 50 µM
p-amidinophenylmethanesulfonyl fluoride hydrochloride, pH
7.1) in a total volume of 80 µl. For competition assay, the binding
reaction was carried out in the presence of a 200-fold molar excess of
unlabeled probe added simultaneously with the labeled probe to the
reaction mixture. For all assays, the protein was added last, and the
reaction mixture was incubated at room temperature for 30 min. The
reaction mixtures were resolved by nondenaturing electrophoresis
through 5% polyacrylamide gels in running buffer (45 mM
Tris borate, pH 7.8). Gels were dried and analyzed with a BAS2000
imaging analyzer (FUJI Film).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of human LUN
cDNA and its deduced amino acid sequence. A, the
complete nucleotide sequence is shown along with the corresponding
translation of the deduced open reading frame. The region (nt
123-317), which is excluded by alternative splicing, is indicated by
square brackets. The first boxed region
corresponds to the RING finger motif, in which the conserved residues
are circled. The region that is likely to adopt a leucine
zipper and a coiled-coil structure is boxed with a
dashed line in which hydrophobic amino acids occurring at
the first position of the heptad repeat are indicated by italic
letters and are dotted. The PEST sequences are
underlined, putative bipartite nuclear localization signals
are indicated by a dashed line, the stop codon is shown by
an asterisk, and the polyadenylation signal is
double-underlined. Two putative SUMO-1 conjugation sites
( KxE) are boxed. The nucleotide sequences have been
deposited in the GenBankTM data base under the accession
numbers AB045732 and AB045733. B, schematic structure of the
LUN protein. The amino acids are numbered below the box. The
closed box corresponds to the RING finger (RF),
which may form a Zn finger-like structure in LUN. The region that is
likely to adopt a leucine zipper (LZ) and a coiled-coil
structure (CC) is represented by a hatched box.
The regions of PEST sequence are dotted, and the nuclear
localization signal (NLS) is indicated by a striped
hatched box. C, amino acid sequence alignments
of the RING finger domains of LUN and other RING finger proteins. The
positions of amino acid residues highly conserved in the family of RING
finger proteins are highlighted with
stippling.
KxE) (27, 28), which is modified in RanGAP1, I
B
, PML,
and p53, was found in the C-terminal half of the protein. The
C-terminal regions (aa 456-731 and 456-882) were reported to interact
with p53 (25) and topoisomerase I (26), respectively.
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Fig. 2.
Chromosome mapping of the LUN
gene to chromosome 9p21 by FISH. A,
metaphase spread hybridized with the 3549-bp (nt 270-3818) probe of
LUN cDNA (arrow). B, the G-banding pattern of
the same metaphase spread as seen in A. C and D,
magnified micrographs of the FISH signals (C) and G-banding
(D) on chromosome 9. The same probes were used as described
in A. E, G-banded ideogram of chromosome 9. Each
dot represents the twin-spot signal detected on chromosome
9. Comparison with the G-banding pattern resulted in assignment of the
LUN gene to chromosome 9p21.
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Fig. 3.
Northern blotting analysis of human LUN
mRNA. Several human tissues were analyzed by Northern blotting
as described under "Experimental Procedures." Each lane
contained ~2 µg of poly(A)+ RNA. The blots were
hybridized with a 3549-bp probe (nt 270-3818) of the LUN cDNA. An
arrow indicates the position of LUN mRNA of 3.8 or 3.6 kb due to alternative splicing. The positions of size markers (in kb)
are indicated on the left.
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Fig. 4.
Localization of LUN transcript in adult lung
tissue. Sections of human lung tissue were hybridized with
antisense (panels A and B) or sense
(panel C) probes. The slides were reacted with the detection
solution for 18 h as described under "Experimental Procedures"
and then observed. Hybridization signals are visible as brown-colored
reaction products. Panel B, higher magnification of
panel A; hybridization signals over a limited number of
squamous cells (type I) and cuboidal cells (type II) appear as brown
products (arrows). Scale bars, 500-µm
(panels A and C) and 200 µm
(panel B).
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Fig. 5.
Subcellular localization of the GFP-LUN
fusion protein in HeLa cells. HeLa cells were transiently
transfected with a mammalian expression plasmid for
LUN51-1045 fused to GFP, pGF- LUN51-1045
(panels A, C, and E) or a control
plasmid expressing GFP, pGF (panels B,
D, and F). 48 h after DNA-transfection,
cells were fixed, illuminated, and observed under a fluorescence
microscope (panels A and B). The same
cells were also viewed by phase-contrast microscopy (panels
C and D). Panels E and F show
digitally merged fluorescent and phase contrast images. Magnification,
×400.
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Fig. 6.
Expression and purification of GST fusion
proteins. Bacterially expressed GST fusion proteins were purified
by glutathione-Sepharose 4B. The purified GST-LUN51-1045
(lane 2), GST-LUN51-374
(lane 3), and GST (lane 1)
were subjected to SDS-polyacrylamide gel electrophoresis and detected
by Coomassie Brilliant Blue R-250 staining. Arrows indicate
the expected positions for the purified proteins. Lane
M contains molecular size standards.
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Fig. 7.
DNA binding characteristics of LUN.
125I-Labeled GST-LUN51-1045,
GST-LUN51-374, and GST proteins (6 × 105
cpm/2 pmol each) were incubated with calf thymus double-stranded DNA
(dsDNA)-cellulose (A) or single-stranded DNA
(ssDNA)-cellulose (B) in binding buffer A (10 mM HEPES-NaOH, 50 mM KCl, 1 mM
2-mercaptoethanol, 20% glycerol, pH 7.1) in the presence or absence of
mixed divalent cations M2+ (1 mM
ZnCl2, 1 mM MgCl2, 1 mM
MnCl2, and 1 mM CaCl2) at 4 °C
for 1 h. After washing with the same buffer, the DNA-bound
radioactivity was determined by -counting. Data are shown as the
means ± S.E. of duplicate determinations.
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Fig. 8.
Effects of divalent cations on LUN-DNA
binding. 125I-Labeled GST-LUN51-1045 was
incubated with double-stranded DNA (dsDNA)-cellulose (A) or
single-stranded DNA (dsDNA)-cellulose (B) in
binding buffer A (10 mM HEPES-NaOH, 50 mM KCl,
1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1) in the
presence of 1 mM ZnCl2, 1 mM
MgCl2, 1 mM MnCl2, 1 mM
CaCl2, and/or 1 mM EDTA alone or in combination
at 4 °C for 1 h as described in Fig. 7. After washing, the
remaining radioactivity on the DNA-cellulose was determined by
-counting. Data are shown as means ± S.E. of duplicate
experiments.
1934 bp from the transcription initiation site, nt 428-460 of GenBankTM D49685) within
the upstream transcriptional regulatory region of the human E-cadherin
gene (39) and two intervening regions (nt 3182-3208 and 5988-6013 of
GenBankTM AF178081) of the human talin gene
exons 2 and 8.
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Fig. 9.
Definition of a consensus binding site for
LUN. Fifty-five binding sequences for LUN obtained by three rounds
of selection are shown. According to alignments, these sequences were
categorized into three groups (A, 25 clones; B,
22 clones; C, 8 clones). The core sequences are indicated in
boxes. Nucleotides in boldface represent the
identity of a given position. Deletions are shown by gaps in the
sequence. The boxed sequence indicates the
palindrome-like binding consensus for LUN. To be considered the
consensus, the threshold level of representation of a nucleotide at a
given position was arbitrary set at >60%. In the case of deletions,
adjacent fixed nucleotides next in sequence were considered the
selected nucleotides.
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Fig. 10.
Gel mobility shift analysis of the binding
consensus for LUN. Identical amounts (8 pmol) of GST
(panel A), GST-LUN51-374
(panel B), or GST-LUN51-1045
(panel C) were incubated with
32P-labeled 31-bp C1 probe (corresponding to the binding
consensus for LUN; 4 × 104 cpm) in the presence or
absence of a 200-fold excess of the indicated double-stranded DNA
competitors (lanes 2-7). Lane
1 shows the binding reaction without fusion proteins.
Free and LUN- complexed DNA fragments were separated on 5%
nondenaturing polyacrylamide gels as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1934 bp from the transcriptional initiation site)
of the human E-cadherin gene (39) and two intervening regions (nt
3182-3208 and 5988-6013) of the human talin gene exons 2 and 8. E-cadherin is a Ca2+-dependent
intercellular adhesion molecule and is closely associated with cancer
metastasis and invasion (39, 42-44). Talin is a cytoskeletal protein
that interacts with extracellular matrix components such as integrin to
form focal adhesion (45, 46). Thus, LUN was proposed to be an
important trans-acting transcriptional regulator of
E-cadherin and talin genes in the alveolar epithelial
cells during lung development, differentiation, and tumorigenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Kazuko Wakai for technical assistance and Yuji Hara and Masayoshi Minakuchi for helpful discussions.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas (to Y. A.) and Scientific Research B (to K. U.) from the Ministry of Education, Science, and Culture, Japan, by a grant from the Naito Foundation (to Y. A.), and by a grant from the Uehara Memorial Foundation (to Y. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB045732 and AB045733.
** To whom correspondence and reprint requests should be addressed. Tel.: 81-774-38-3222; Fax: 81-774-38-3226; E-mail: adachi@ scl.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010262200
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ABBREVIATIONS |
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The abbreviations used are: RING, really interesting new gene; kb, kilobase pair(s); bp, base pair(s) PCR, polymerase chain reaction; nt, nucleotides; aa, amino acids; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; GST, glutathione S-transferase; SUMO, small ubiquitin-related modifier; SCLC, small cell lung cancer.
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---|
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---|
1. | Freemont, P. S. (1993) Ann. N. Y. Acad. Sci. 684, 174-192[Medline] [Order article via Infotrieve] |
2. | Saurin, A. J., Borden, K. L. B., Boddy, M. N., and Freemont, P. S. (1996) Trends Biochem. Sci. 21, 208-214[CrossRef][Medline] [Order article via Infotrieve] |
3. | Barlow, P. N., Luisi, B., Milner, A., Elliott, M., and Everett, R. (1994) J. Mol. Biol. 237, 201-211[CrossRef][Medline] [Order article via Infotrieve] |
4. | Everett, R. D., Barlow, P., Milner, A., Luisi, B., Orr, A., Hope, G., and Lyon, D. (1993) J. Mol. Biol. 234, 1038-1047[CrossRef][Medline] [Order article via Infotrieve] |
5. | Borden, K. L. B., Boddy, M. N., Lally, J., O'Reilly, N. J., Martin, S., Howe, K., Solomon, E., and Freemont, P. S. (1995) EMBO J. 14, 1532-1541[Abstract] |
6. |
Tagawa, M.,
Sakamoto, T.,
Shigemoto, K.,
Matsubara, H.,
Tamura, Y.,
Ito, T.,
Nakamura, I.,
Okitsu, A.,
Imai, K.,
and Taniguchi, M.
(1990)
J. Biol. Chem.
265,
20021-20026 |
7. | Kanno, M., Hasegawa, M., Ishida, A., Isono, K., and Taniguchi, M. (1995) EMBO J. 14, 5672-5678[Abstract] |
8. | Lovering, R., Hanson, I. M., Borden, K. L. B., Martin, S., O'Reilly, N. J., Evan, G. I., Rahman, D., Pappin, D. J. C., Trowsdale, J., and Freemont, P. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2112-2116[Abstract] |
9. |
Joazeiro, C. A.,
Wing, S. S.,
Huang, H.,
Leverson, J. D.,
Hunter, T.,
and Liu, Y. C.
(1999)
Science
286,
309-312 |
10. |
Lorick, K. L.,
Jensen, J. P.,
Fang, S.,
Ong, A. M.,
Hatakeyama, S.,
and Weissman, A. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11364-11369 |
11. | Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., Tavtigian, S., Liu, Q., Cochran, C., Bennett, L. M., Ding, W., Bell, L., Rosenthal, J., Hussey, C., Tran, T., McClure, M., Frye, C., Hattier, T., Phelps, R., Haugen-Strano, A., Katcher, H., Yakumo, K., Gholami, Z., Shaffer, D., Stone, S., Bayer, S., Wray, C., Bogden, R., Dayananth, P., Ward, J., Tonin, P., Narod, S., Bristow, P. K., Norris, F. H., Helvering, L., Morrison, P., Rosteck, P., Lai, M., Barrett, J. C., Lewis, C., Neuhausen, S., Cannon-Albright, L., Goldgar, D., Wiseman, R., Kamb, A., and Skolnick, M. H. (1994) Science 266, 66-71[Medline] [Order article via Infotrieve] |
12. | Goddard, A. D., Borrow, J., Freemont, P. S., and Solomon, E. (1991) Science 254, 1371-1374[Medline] [Order article via Infotrieve] |
13. | Kakizuka, A., Miller, W. H., Jr., Umesono, K., Warrell, R. P., Jr., Frankel, S. R., Murty, V. V. V. S., Dmitrovsky, E., and Evans, R. M. (1991) Cell 66, 663-674[Medline] [Order article via Infotrieve] |
14. | Hasegawa, N., Iwashita, T., Asai, N., Murakami, H., Iwata, Y., Isomura, T., Goto, H., Hayakawa, T., and Takahashi, M. (1996) Biochem. Biophys. Res. Commun. 225, 627-631[CrossRef][Medline] [Order article via Infotrieve] |
15. | Blake, T. J., Heath, K. G., and Langdon, W. Y. (1993) EMBO J. 12, 2017-2026[Abstract] |
16. | von Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H., and Berns, A. (1991) Cell 65, 737-752[Medline] [Order article via Infotrieve] |
17. | Haupt, Y., Alexander, W. S., Barri, G., Peter Klinken, S., and Adams, J. M. (1991) Cell 65, 753-763[Medline] [Order article via Infotrieve] |
18. | Goebl, M. G. (1991) Cell 66, 623[Medline] [Order article via Infotrieve] |
19. | Takahashi, H., Behbakht, K., McGovern, P. E., Chiu, H. C., Couch, F. J., Weber, B. L., Friedman, L. S., King, M. C., Furusato, M., LiVolsi, V. A., Menzin, A. W., Liu, P. C., Benjamin, I., Morgan, M. A., King, S. A., Rebane, B. A., Cardonick, A., Mikuta, J. J., Rubin, S. C., and Boyd, J. (1995) Cancer Res. 55, 2998-3002[Abstract] |
20. | Inazawa, J., Sasaki, H., Nagura, K., Kakazu, N., Abe, T., and Sasaki, T. (1996) Hum. Genet. 98, 508-510[CrossRef][Medline] [Order article via Infotrieve] |
21. | Inazawa, J., Ariyama, T., Abe, T., Druck, T., Ohta, M., Huebner, K., Yanagisawa, J., Reed, J. C., and Sato, T. (1996) Genomics 31, 240-242[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Adachi, Y.,
Kitahara-Ozawa, A.,
Sugamura, K.,
Lee, W. J.,
Yodoi, J.,
Maki, M.,
Murachi, T.,
and Hatanaka, M.
(1992)
J. Biol. Chem.
267,
19373-19378 |
23. | Morimoto, M., Morita, N., Ozawa, H., Yokoyama, K., and Kawata, M. (1996) Neurosci. Res. 26, 235-269[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract] |
25. | Zhou, R., Wen, H., and Ao, S. Z. (1999) Gene 235, 93-101[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Haluska, P., Jr.,
Saleem, A.,
Rasheed, Z.,
Ahmed, F.,
Su, E. W.,
Liu, L. F.,
and Rubin, E. H.
(1999)
Nucleic Acids Res.
27,
2538-2544 |
27. |
Rodriguez, M. S.,
Desterro, J. M. P.,
Lain, S.,
Midgley, C. A.,
Lane, D. P.,
and Hay, R. T.
(1999)
EMBO J.
18,
6455-6461 |
28. | Desterro, J. M. P., Rodriguez, M. S., and Hay, R. T. (1998) Mol. Cell 2, 233-239[Medline] [Order article via Infotrieve] |
29. | Kastner, P., Perez, A., Lutz, Y., Rochette-Egly, C., Gaub, M. P., Durand, B., Lanotte, M., Berger, R., and Chambon, P. (1992) EMBO J. 11, 629-642[Abstract] |
30. | Patarca, R., Schwartz, J., Singh, R. P., Kong, Q. T., Murphy, E., Anderson, Y., Sheng, F. Y., Singh, P., Johnson, K. A., Guarnagia, S. M., Durfee, T., Blattner, F., and Cantor, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2733-2737[Abstract] |
31. | Everett, R. D., and Maul, G. G. (1994) EMBO J. 31, 5062-5069 |
32. | Spanopoulou, E., Zaitseva, F., Wang, F. H., Santagata, S., Baltimore, D., and Panayotou, G. (1996) Cell 87, 263-276[Medline] [Order article via Infotrieve] |
33. |
Mo, X.,
Bailin, T.,
and Sadofsky, M. J.
(1999)
J. Biol. Chem.
274,
7025-7031 |
34. | Lo, K., Landau, N. R., and Smale, S. T. (1991) Mol. Cell. Biol. 11, 5229-5243[Medline] [Order article via Infotrieve] |
35. | Molnar, A., and Georgopoulos, K. (1994) Mol. Cell. Biol. 14, 8292-8303[Abstract] |
36. | Driever, W., and Nusslein-Volhard, C. (1989) Nature 337, 138-143[CrossRef][Medline] [Order article via Infotrieve] |
37. | Driever, W., Thoma, G., and Nusslein-Volhard, C. (1989) Nature 340, 363-367[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chen, Y., and Gill, G. N. (1996) J. Biol. Chem. 271, 5181-5188 |
39. | Yoshiura, K., Kanai, Y., Ochiai, A., Shimoyama, Y., Sugimura, T., and Hirohashi, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7416-7419[Abstract] |
40. | Jones, J. S., Weber, S., and Prakash, L. (1988) Nucleic Acids Res. 16, 7119-7131[Medline] [Order article via Infotrieve] |
41. | Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989) Cell 59, 1035-1048[Medline] [Order article via Infotrieve] |
42. | Christofori, G., and Semb, H. (1999) Trends Biochem. Sci. 24, 73-76[CrossRef][Medline] [Order article via Infotrieve] |
43. | Sommers, C. L., Thompson, E. W., Torri, J. A., Kemler, R., Gelmann, E. P., and Byers, S. W. (1991) Cell Growth Differ. 2, 365-372[Abstract] |
44. | Oka, H., Shiozaki, H., Kobayashi, K., Inoue, M., Tahara, H., Kobayashi, T., Takatsuka, Y., Mastuyoshi, N., Hirano, S., Takeichi, M., and Mori, T. (1993) Cancer Res. 53, 1696-1701[Abstract] |
45. | Goldmann, W. H. (2000) Biochem. Biophys. Res. Commun. 271, 553-557[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Albiges-Rizo, C.,
Frachet, P.,
and Block, M. R.
(1995)
J. Cell Sci.
108,
3317-3329 |
47. | Blank, V., and Andrews, N. C. (1997) Trends Biochem. Sci. 22, 437-441[CrossRef][Medline] [Order article via Infotrieve] |
48. | Kataoka, K., Noda, M., and Nishizawa, M. (1994) Mol. Cell. Biol. 14, 700-712[Abstract] |
49. | Glover, J. N. M., and Harrison, S. C. (1995) Nature 373, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
50. | Ellenberger, T. E., Brandl, C. J., Struhl, K., and Harrison, S. C. (1992) Cell 71, 1223-1237[Medline] [Order article via Infotrieve] |
51. |
Mao, Y.,
Sun, M.,
Desai, S. D.,
and Liu, L. F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4046-4051 |
52. | Kim, S. K., Ro, J. Y., Kemp, B. L., Lee, J. S., Kwon, T. J., Fong, K. M., Sekido, Y., Minna, J. D., Hong, W. K., and Mao, L. (1997) Cancer Res. 57, 400-403[Abstract] |
53. | Bohm, M., Totzeck, B., and Wieland, I. (1994) Clin. Exp. Metastasis 12, 55-62[Medline] [Order article via Infotrieve] |