From the Institut Fédératif de Recherche Claude de Préval, Université Paul Sabatier and Centre Hospitalo-Universitaire de Toulouse, INSERM Unité 326, Phospholipides Membranaires, Signalisation Cellulaire et Lipoprotéines, Hôpital Purpan, F 31059 Toulouse Cedex, France
Received for publication, October 26, 2000, and in revised form, February 6, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enterocyte terminal differentiation occurs at the
crypt-villus junction through the transcriptional activation of
cell-specific genes, many of which code for proteins of the brush
border membrane such as intestinal alkaline phosphatase,
sucrase-isomaltase, or the microvillar structural protein villin.
Several studies have shown that this sharp increase in specific
mRNA levels is intimately associated with arrest of cell
proliferation. We isolated several clones from a guinea pig intestine
cDNA library. They encode new proteins characterized by an original
structure associating a carboxyl-terminal B30.2/RFP-like domain and a
long leucine zipper at the amino terminus. The first member of this
novel gene family codes for a 65-kDa protein termed enterophilin-1,
which is specifically expressed in enterocytes before their final
differentiation. Enterophilin-1 is the most abundant in the small
intestine but is still present in significant amounts in colonic
enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized
by a specific anti-enterophilin-1 antibody, and its expression was
positively correlated with cell differentiation status. In addition,
transfection of HT-29 cells with enterophilin-1 full-length cDNA
slightly inhibited cell growth and promoted an increase in alkaline
phosphatase activity. Taken together, these data identify enterophilins
as a new family of proteins associated with enterocyte differentiation.
The self-renewing small intestinal epithelium represents an
attractive and valuable system to study various processes occurring during cell life such as proliferation, differentiation, or apoptosis. Proliferation is limited to the crypts of Lieberkühn, where
multipotent stem cells achieve continuous renewal of four main
epithelial lineages. At the top of the crypt, cells lose their
proliferative ability and complete differentiation during a highly
organized migration along the crypt-villus axis. Enterocytes,
mucus-producing goblet cells, and enteroendocrine cells evolve during a
vertical migration to the villus apex (1-3). This process requires 4 to 5 days and results in well-differentiated cells that are finally released into the intestinal lumen upon programmed cell death. In
contrast, Paneth cells undergo terminal differentiation while moving
down to the base of the crypt (4-8). The differentiation process and
the function of the epithelium also vary along the horizontal axis
(from proximal to distal intestine), and in both cases, functional
differences reflect various patterns of gene expression as well as the
nature of the epithelial cell type (7).
How these various events are regulated at the genomic level and the
intracellular signaling pathways involved in this differentiation process are still poorly understood. Recently identified genes were
found to be necessary either to maintain the proliferative status of
stem cells (Tcf-4) (9) or to direct the differentiation process
(homeobox transcription factors cdx1 and cdx2) (10-12). Most of the
previous studies were performed with enterocytes (95% of
differentiated cells of the villus) and have essentially described the
specific expression of genes contributing to the morphological and
functional phenotype of mature cells. For instance, villin participates
in the formation of the typical cytoskeleton underlying the
microvillosities of absorptive cells, whereas various brush border
membrane hydrolases are involved in the digestive function of
enterocytes. These include alkaline phosphatase, aminopeptidase N,
sucrase-isomaltase (13-15), or phospholipase B, also called AdRabB
(16-19). The promoter of the sucrase-isomaltase gene has been studied
extensively to delineate the molecular mechanisms regulating the
expression of genes involved in enterocyte differentiation (20).
Interestingly, a common structure for enterocyte-specific promoters has
been suggested for the sucrase-isomaltase and phospholipase B genes
(21).
During our first attempts to clone phospholipase B
cDNA, we isolated various clones encoding new proteins containing a
B30.2 domain (22, 23). The B30.2 domain is a 160-170-amino acid globular domain present at the carboxyl-terminal half of a rapidly growing number of proteins classified according to the nature of their
amino-terminal part (24-26). The main category consists of nuclear
proteins with a B box and a RING motif, such as RFP (27-29),
Sjögren syndrom type-A or Ro/SS-A antigen (30), and Xenopus
laevis nuclear factor Xnf7 (31). The importance of these proteins
was illustrated by the discovery of two genes, MEFV and MID1, whose mutations in the region encoding the B30.2
domain appear to be responsible for two genetic deficiencies, familial Mediterranean fever (32, 33) and Opitz G/BBB syndrome (34), respectively. Unlike other RING finger proteins, the MID1
product is not found in the nucleus but is associated with microtubules (35), and a conserved fibronectin type III domain was identified in
the protein (36).
Butyrophilin, a membrane protein expressed in milk fat globule
membrane, is the prototype of a second group of proteins whose intracellular B30.2 domain is linked to two external
immunoglobulin-like motifs (IgV-IgC1) by a single transmembrane segment
(37-39). Recently, a gene sharing high homology with butyrophilin and
encoding a new protein called erythroid membrane-associated protein
(ERMAP) was described (40). A third group consists of extracellular proteins such as the We now describe a new group of intestinal proteins bearing a
carboxyl-terminal B30.2 domain and an extended leucine zipper containing up to 45 regular heptad repeats in their amino terminus. One
member of this novel gene family codes for a 65-kDa protein termed
enterophilin-1. Both the endogenous expression pattern and transfection
experiments reported herein indicate a close relationship between
enterophilin-1 expression and enterocyte differentiation.
Tissue and Cells--
The entire small intestine was removed and
excised into fragments corresponding to duodenum, jejunum, ileum, and
colon for regional dissection. The whole ileum was used, the duodenum
was cut into two fragments (proximal and distal), and the jejunum and
colon were divided into three equal segments (proximal, middle, and
distal). Mucosa was scraped and frozen in liquid nitrogen for protein
analysis. Intestinal epithelial cells were isolated and separated as a
villus to crypt gradient according to Weiser (42). COS-7, HT-29, and
Caco-2 cells (American Type Culture Collection) were grown in
Dulbecco's modified Eagle's medium with glucose as a carbon source,
supplemented with 2 mM glutamine, 10% fetal bovine serum,
and 100 µg/ml penicillin/streptomycin in a humidified atmosphere
containing 5% CO2. Fetal bovine serum was increased up to
20%, and nonessential amino acids (0.1 mM) were added to
the culture medium of Caco-2 cells.
RNA Isolation--
Total RNA was extracted from different
tissues or from isolated intestinal cells by the cesium
chloride/guanidinium isothiocyanate method (43). Polyadenylated RNA was
prepared using oligo(dT) cellulose columns (Amersham Pharmacia Biotech).
cDNA Library Construction and Screening--
Guinea pig
intestinal cDNA library was constructed in
For immunological screening, the nitrocellulose filters were probed
with anti-phospholipase B antiserum (18) and then probed with an
anti-rabbit immunoglobulin-G antibody conjugated with alkaline
phosphatase. Positive plaques were purified by three runs and recovered
as Bluescript plasmids by in vivo excision using ExAssist
helper phage (Stratagene).
For DNA screening, the library was plated on XL1-Blue MRF' E. coli, and replicas on nylon membranes (Hybond N+;
Amersham Pharmacia Biotech) were prepared. Phage DNA was denatured (1.5 M NaCl, 0.5 M NaOH) and cross-linked to the
membrane by exposure to UV. The 9D1 PstI fragment was used
as a probe for hybridization as described previously (19). Positive
clones were purified through three runs and recovered as Bluescript
plasmids by in vivo excision using ExAssist helper phage and
SOLR E. coli (Stratagene).
Radioactive Probes and Northern Blots--
Enterophilin-1 probe
(850 bp1) was obtained by
digestion of the 3.9D1 clone with EcoRI/XhoI.
Phospholipase B probe is a 386-bp fragment amplified by polymerase
chain reaction (19). The glyceraldehyde-3-phosphate dehydrogenase probe
(350 bp) was prepared by polymerase chain reaction using human lung
cDNA as a template (44). The probes were labeled by random primer
extension using the Nonaprimer Kit (Appligene Oncor) and
[ Construction of Eukaryotic Expression Vectors--
The
pBluescript-3.9D1 clone containing the full-length cDNA of
enterophilin-1 was used as a template for these constructions. pCI-Ent1
was obtained by digestion of pBluescript-3.9D1 with XbaI and
KpnI, and the insert was ligated in pCI (Promega) previously digested with NheI and KpnI. To generate
pGFP-Ent1 and pcDNA3-Ent1, double digestions were achieved with
PstI/KpnI and NotI/HindIII, and the insert cDNAs were ligated respectively in pGFP-C1
(CLONTECH) and pcDNA3.1/Myc-His (Invitrogen) at
the corresponding sites. We generated enterophilin-1 in frame with the
carboxyl terminus of green fluorescent protein (GFP) and
carboxyl-terminal c-myc and polyhistidine tag.
Transfection and Fluorescence Microscopy--
COS-7 or HT-29
cells were plated in 60-mm dishes or 6-well plates and transiently
transfected with the different plasmids using a cationic lipid
(LipofectAMINE; Life Technologies, Inc.) according to the
manufacturer's protocol. For transfection experiments with the GFP
fusion constructs, COS-7 cells were grown on sterile glass coverslips
placed in 35-mm culture plates and fixed as described previously (45).
Fluorescence was visualized at 65 h with a Zeiss Axioskop
microscope at a magnification of ×40.
Preparation of B30.2 Fusion Protein--
The pBluescript-3.9D1
plasmid was digested with EcoRI and XhoI, and the
obtained fragment (residues 336-529) corresponding to the 3' region of
enterophilin-1 cDNA (B30.2 domain) was inserted into pGEX-KG vector
(a gift of Dr. J. E. Dixon, University of Michigan Medical School,
Ann Arbor, MI). This construction was introduced into E. coli strain XL1-Blue, and production of the fusion protein was
induced with isopropyl-1-thio- Preparation of Anti-B30.2 and Anti-Peptide Polyclonal
Antibodies--
After SDS-polyacrylamide gel electrophoresis under
reducing conditions according to Laemmli (46) and Coomassie Blue
staining, the 55-kDa band corresponding to the GST-B30.2 domain fusion
protein was used to immunize a New Zealand white rabbit. The
antiserum was purified by a two-step procedure, including preadsorption on GST bound to glutathione-agarose beads, followed by adsorption of
the subsequent supernatant on GST-B30.2 fusion protein bound to
glutathione-agarose beads. The specific anti-B30.2 antibody was eluted
by acidification with 0.1 M citric acid, pH 2.7, and immediately neutralized with 1 M Tris-HCl, pH 11.0. The
purified antibody was desalted and used for immunoblotting.
A rabbit polyclonal anti-peptide antibody was produced by Eurogentec
(Seraing, Belgium) using a peptide of 16 residues (CQTERDKLRQEIDDRK) corresponding to amino acids 313-328 of enterophilin-1 (Fig. 1), where
the amino-terminal leucine was replaced by a cysteine to allow
conjugation to the keyhole limpet hemocyanin carrier.
Preparation of Cell Extracts and Immunoblotting--
All the
procedures described were performed at 4 °C. Nine fractions of
guinea pig enterocytes were isolated according to Weiser (42), washed
twice in PBS containing 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 2 mM leupeptin, and then disrupted by 10 strokes with a
Potter homogenizer. Caco-2 cells were harvested with trypsin/EDTA
either at confluence (mainly undifferentiated cells) or at various
times (up to 23 days) after reaching confluence (differentiated cells).
They were washed twice in PBS and resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, and 2 mM leupeptin).
Nuclei were isolated as described previously (47). Briefly, washed
cells (about 108 cells) were gently suspended in hypotonic
buffer containing 0.5% Nonidet P-40. They were incubated on ice for 10 min, and nuclei were pelleted by centrifugation for 5 min at 1,000 × g. They were then washed twice with hypotonic buffer
without detergent and then suspended in the same buffer.
Protein concentration was determined according to the method of
Bradford (48). Protein samples were submitted to SDS-polyacrylamide gel
electrophoresis (46) and transferred onto nitrocellulose membranes
according to standard protocol (49).
Monoclonal antibody against [3H]Thymidine Incorporation--
HT-29
cells were transfected as described previously and maintained in the
presence of serum. [3H]Thymidine (0.5 µCi/ml) was added
to the cells at 24, 48, or 72 h after transfection, and incubation
was prolonged for 4 h. Cells were washed twice in PBS, and 5%
trichloroacetic acid was added for 10 min at room temperature. The
solution was withdrawn, and solubilization was performed at room
temperature with 0.5 M NaOH for at least 30 min.
Radioactivity in cell lysates was measured by liquid scintillation
counting (1900 TR; Packard).
Alkaline Phosphatase and Sucrase Activities--
Caco-2 or
transfected HT-29 cells were scraped in PBS and sonicated, and the
homogenates were incubated in the presence of saccharose or
p-nitrophenyl phosphate as substrates according to the
instructions of the manufacturer (Roche Molecular Biochemical). Data
are expressed in IU (micromole of substrate hydrolyzed per minute) per
gram of protein. Cell proteins were determined according to the method
of Bradford (48).
Isolation of cDNA Coding for Enterophilins--
A
As also indicated in Fig. 1, the amino-terminal part of enterophilin-1
(residues 11-326) contained 45 heptad repeats preceded by a leucine
residue (32 repeats), an aromatic amino acid (3 repeats), or a basic
residue (7 histidines, 2 lysines, and 1 arginine). This sequence
did not reveal significant homology with any known protein.
Besides five other clones related to enterophilin-1 but differing by
their amino-terminal end, the seventh clone (7.9D1) coded for a
putative protein containing 18 heptad repeats. However, despite the
presence of the whole B30.2 domain, a frameshift introduced a premature
stop codon limiting the protein sequence to the leucine zipper domain.
As shown in Fig. 1, the corresponding protein was referred to as
enterophilin-2-short (Ent-2-S). Finally, the sequence of the 9D1 clone
perfectly matched the sequence of Ent-2-S but still contained the
intact B.30.2 domain. This was referred to as enterophilin-2-long
(Ent-2-L), but its sequence is not presented herein because it did not
contain the methionine initiation codon.
These sequence data indicated that enterophilins could be the
prototypes of a new subgroup among the proteins bearing a B30.2 domain.
Their most remarkable feature is a very long leucine zipper. As
illustrated in Fig. 2A, this
allowed the prediction of an almost perfect geometrical alignment of
amino acid residues at each position of the Subcellular Localization of Enterophilin-1--
Using the
anti-peptide antibody described under "Experimental Procedures,"
enterophilin-1 was detected as a 65-kDa protein in isolated guinea pig
enterocytes (Fig. 3C). This
corresponded to its predicted molecular mass, indicating that
enterophilin-1 probably did not undergo any posttranslational
modification. These data were confirmed by in vitro coupled
transcription translation with the pCI-Ent-1 construct (Fig.
3A), and they fitted with the detection of an identical
polypeptide in pCI-Ent-1-transfected COS-7 cells (Fig. 3B).
Moreover, following an efficient fractionation of enterocytes, as shown
by the segregation of villin from lamin B, enterophilin-1 was found to
be concentrated in the nuclear fraction, although some amounts could
still be detected in the cytosol (Fig. 3C).
Further attempts to localize enterophilin-1 by immunohistochemical
techniques remained unsuccessful, probably because the epitopes
recognized by both the anti-peptide and the anti-B30.2 domain
antibodies remained buried either in the protein itself or by
associated proteins. Thus, indirect evidence for a nuclear localization
of enterophilin-1 was obtained using COS-7 cells transfected with
pGFP-Ent-1, allowing the expression of enterophilin-1 in fusion with
GFP. As shown in Fig. 3D, 1, fluorescence microscopy analysis showed that cells transfected with pGFP-Ent-1 displayed a
diffuse cytosolic staining, whereas a strong nuclear signal could be
observed. In contrast, GFP alone was distributed throughout the cell
(Fig. 3D, 3). Identical results were obtained using
pcDNA3-Ent-1 for cell transfection, with the carboxyl-terminal
c-myc end being detected with an anti-c-myc antibody (data not shown).
Northern Blot Analysis of Enterophilin-1 mRNA Tissue
Distribution--
A number of guinea pig tissues were analyzed by
Northern blotting to detect the presence of enterophilin-1
mRNA (Fig. 4). A large 2-kb
transcript was highly expressed in intestine and slightly detected in
heart. Moreover, a weak band corresponding to a larger mRNA was
observed in the lung. The smeared signal detected in the intestine
suggested a possible RNA degradation. However, rRNA staining (Fig. 4,
bottom panel) and glyceraldehyde-3-phosphate dehydrogenase
probe hybridization signal (Fig. 4, middle panel), which
were used to compare RNA loading, suggested that this was not the case.
The smeared signal probably results from the diversity of enterophilin
mRNA, as already suggested by data from cDNA cloning. In
support of this, Northern blotting performed with 9D1 as a probe (which
codes for Ent-2-L; see above) resulted in a similar smeared signal
(data not shown).
Expression of Enterophilin-1 in Guinea Pig Intestine--
The
intestine can be structurally and functionally divided into several
different regions, namely the duodenum, jejunum, ileum, and proximal
and distal colon. Previous studies have shown differences in the
expression of several genes along the intestine, reflecting this
functional diversity (7). As shown by Western blotting experiments, the
highest expression level of enterophilin-1 was observed in the proximal
part of the intestine (duodenum and jejunum) and then declined in the
ileum to remain at a significantly lower level in the different
segments of colon (Fig.
5A).
Experiments were also performed to define the expression of
enterophilin-1 along the crypt-villus axis. Guinea pig enterocytes were
isolated at different stages of differentiation according to Weiser
(42). As shown in Fig. 5B, enterophilin-1 was expressed in
all fractions, with an increased level in fractions 5 and 6, whereas it
declined in the most differentiated enterocytes (fractions 1-3). These
data suggest that enterophilin-1 is expressed at the early stages of
the differentiation process and that its expression level is increased
when cells undergo terminal differentiation.
Enterophilin-1 Expression Is Related to Enterocyte Terminal
Differentiation--
Using the anti-peptide and the anti-B30.2
antibodies, we could identify in Caco-2 cells an immunoreactive 65-kDa
protein displaying the same subcellular localization as guinea pig
intestinal enterophilin-1 (data not shown). Although we have no
definitive proof that this protein is the human orthologue of
enterophilin-1, we decided to study its expression pattern during
Caco-2 cell differentiation (51). Fig.
6A shows a typical growth
curve of Caco-2 cells. Proliferation began after a lag time of 2 days,
cells reached confluence after 7 days in culture, and then growth
leveled off between 9 and 12 days. At confluence, domes characteristic
of functional differentiation appeared all over the monolayer as described previously (52, 53). We confirmed this observation by
following the expression of sucrase-isomaltase and alkaline phosphatase, two well-known intestinal brush border enzymes used as
differentiation markers. As shown in Fig. 6C, both hydrolase activities were low during the proliferative phase and increased when
growth reached a plateau (12 days). In parallel, Western blot
analysis showed that enterophilin-1-related protein displayed an
increased expression beginning slightly before (9 days) that of
sucrase-isomaltase and alkaline phosphatase, with a high level of
expression being maintained during the stationary phase (Fig. 6,
B and C).
Upon adding butyrate to the culture, Caco-2 cells started
differentiation in a more coordinated way than by confluence-induced differentiation. Western blot analysis of enterophilin-1-related protein expression showed a sharp increase 1 day after butyrate addition (Fig. 7). An increase in
alkaline phosphatase activity was also observed, but the peak was
delayed (4 days after induction). Sucrase activity was also induced,
but to a lower extent.
Finally, to explore the possible functional role of enterophilin-1 in
enterocyte differentiation, undifferentiated HT-29 cells were
transiently transfected with pcDNA3-Ent1 and analyzed for cell
proliferation and differentiation. DNA synthesis was estimated by
[3H]thymidine incorporation, and data in Fig.
8A indicate that
enterophilin-1-transfected cells exhibited a slower growth rate than
cells transfected with the vector alone. This resulted in a 30%
decrease in [3H]thymidine incorporation 48 h after
cell transfection. A slight reduction of cell number was also observed
at 72 h in enterophilin-1-transfected cells compared with control
cells (Fig. 8B). Finally, 72 h after transfection, a
2.5-fold increase in alkaline phosphatase activity was observed in
cells transfected with enterophilin-1 cDNA compared with those
transfected with vector alone (Fig. 8C). No alkaline phosphatase activity could be detected after 24 or 48 h under our
experimental conditions.
Enterophilins, whose cDNA was actually isolated by serendipity
in a phage screen using anti-phospholipase B serum, appear to be a new
subfamily of proteins with a B30.2 domain. Their structure is
particularly interesting because of the presence of an unusually long
and regular leucine zipper. Long leucine zippers are detected in
proteins of the bZIP superfamily such as murine LZIP-1 and LZIP-2 (54),
their human homologue Luman (55), hMAF (human MAF) (56), the
light-regulated transcription factor ATB2 from Arabidopsis
thaliana (57), or the zipper protein isolated from the brush
border membrane of chicken intestine, which regulates actin/myosin 1 interactions (58, 59). However, the latter one contains only 27 heptad
repeats that do not display the same regularity as enterophilins.
The large number of heptad repeats in enterophilins suggests that these
proteins could be able to homo- or heterodimerize. Indeed, sequence
analysis of enterophilin-1 using "Prosite" allows us to predict two
coiled coil regions corresponding to residues 10-15 and 126-334.
However, only enterophilin-1 displays neutral or hydrophobic residues
at position d of the The recently described signaling domain SPRY, which is a subdomain of
the B30.2 region (23, 67), is also present in enterophilin-1 between
position 403 and 523 of the amino acid sequence. The SPRY domain was
identified in several products of genes involved in severe diseases
such as pyrin/marenostrin and midin, suggesting that enterophilin-1
could be involved in some pathologies.
We have focused our investigations on enterophilin-1, which appeared to
display a nuclear localization. This may be linked to the large number
of basic residues, concentrated at specific positions of the Enterophilin-1 has tissue-restricted expression and appears to be
synthesized mainly in the small intestine. Analysis of Weiser fractions
demonstrated that it is abundant in the early differentiating cells of
the mid-villus region (fractions 5 and 6), where enterocytes progressing along the crypt-villus unit enter their terminal
differentiation program.
Although we have no definitive evidence that Caco-2 cells express the
putative human orthologue of enterophilin-1, the detection of an
enterophilin-1-related protein displaying the same subcellular localization is strongly suggestive of the presence of a very similar
if not identical protein in human bowel. Moreover, upon in
vitro differentiation of Caco-2 cells, enterophilin-1-related protein displayed an expression pattern that was very reminiscent of
our observations on Weiser fractions. Altogether, these data strongly
suggest that enterophilin-1 is expressed rather early during the
differentiation program, at least slightly before brush border hydrolases.
In addition, enterophilin-1 expression in HT-29 promoted the appearance
of alkaline phosphatase and slightly inhibited cell growth. However, we
cannot conclude from our present data that enterophilin-1 plays a
direct causal role in the late enterocyte differentiation program. The
diversity of enterophilins, as suggested by cDNA cloning as well as
by Northern analysis, might explain why overexpression of
enterophilin-1 alone is not sufficient to induce a dramatic effect on
cell proliferation. Additional studies will be required to better
depict and understand the observed changes.
The involvement of a protein with a B30 domain in the differentiation
process was illustrated by the work of Harada et
al. (70), demonstrating that mouse hematopoiesis-specific
ring finger protein (HERF-1) is required for terminal differentiation
of erythroid cells. Furthermore, the expression of the
estrogen-responsive finger protein (efp) is regulated during osteoblast
differentiation (71). The ability of these proteins to induce
differentiation could be related to their DNA binding properties,
suggesting a role in transcriptional regulation, as described
previously for efp (72). However, the sequences of the promoters
targeted by these factors remain to be determined.
The familial Mediterranean fever protein (pyrin) is expressed
preferentially in granulocytes and myeloid bone marrow precursors (32,
33), and recent data indicated that its level was enhanced in HL-60
cells undergoing stimulated granulocytic differentiation (73). A very
recent study suggested that at some stage of its functional pathway,
pyrin resides in the cytoplasm and might be related to protein sorting
by the Golgi apparatus (74).
In conclusion, identification of enterophilins adds a new subgroup to
the rapidly expanding family of proteins with a B30.2 domain. Very few
of these proteins have been characterized on a functional point of
view. Additional studies of enterophilins might provide data allowing
us to progress in our understanding of the molecular mechanisms
involved in enterocyte differentiation and colonic neoplasia. Because
of its leucine zipper structure, enterophilin-1 could be part of a
multiprotein complex. Therefore, identification of its cellular
partners as well as the use of loss or gain of function in transgenic
mice might open some promising fields.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of stonustoxins, two
hypotensive and lethal toxins isolated from stonefish (41).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II vector using 5 µg of polyadenylated RNA from differentiated villus enterocytes
according to the instructions of the manufacturer (ZAP cDNA
synthesis kit, Stratagene, La Jolla, CA). The library was packaged into
phage particles using the Gigapack II system (Stratagene) and amplified
once in XL1-Blue MRF' Escherichia coli (Stratagene).
-32P]dCTP from Amersham. RNA electrophoresis and
filter hybridization were performed as described previously (19).
-D-galactopyranoside. The
glutathione S-transferase (GST) fusion protein was purified by affinity chromatography on glutathione-agarose beads (Sigma).
-actin was from Sigma. Anti-villin
antibody was from Affiniti, and anti-lamin B was from Nova Castra Laboratories.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II
guinea pig intestinal cDNA library was constructed and screened
using a polyclonal antibody raised against phospholipase B (18, 19).
Three identical positive clones (7D1, 8D1, and 9D1) carrying
1.5-kb insertions were isolated and sequenced. Because none of them apparently contained an ATG initiation codon, isolation of
the full-length cDNA was attempted using the PstI
1,100-bp fragment of 9D1 as a probe. Seven positive clones were
selected and sequenced. Among them, 3.9D1 corresponded to a complete
nucleotide sequence of 1999 bp. The open reading frame designed
according to Kozak (50) included 1,587 nucleotides (positions
142-1728) and encoded a 64.5-kDa protein consisting of 529 amino
acids. The lack of signal sequence or putative transmembrane domain in the polypeptide suggested an intracellular localization. As shown in
Fig. 1, the carboxyl-terminal region of
the sequence (residues 352-508) was highly identical to the B30.2
domain of butyrophilin (41% identity) (37, 38), and
marenostrin/pyrin (40% identity) (32, 33). Because of its preferential
intestinal expression (see below) and according to its homology with
the B30.2 domain of butyrophilin, the product of the 3.9D1 clone was
named enterophilin-1.
View larger version (61K):
[in a new window]
Fig. 1.
Sequence alignments of guinea pig Ent-1, the
short form of enterophilin-2 (Ent-2S), human
butyrophilin (BT), and marenostrin/pyrin
(M/P). Leucine residues of the heptad repeats are
in bold. Positions with full identity between enterophilins
are indicated by an asterisk above the sequence, whereas + denotes a partial conservation of the following groups: D, E, N,
Q; S, T; Y, F; V, L, I; and R, K, H. The same symbols below
the sequences compare the B30.2 domains of the three proteins. The
sequence comprised between residues 170 and 195 of Ent-2S
(discontinuously underlined), which corresponds to a change
in the open reading frame, is not considered for sequence comparison.
Dashes correspond to gaps allowing maximal alignment.
Typical motifs of B30.2 domain are in bold (LDP, WEV,
LDY). Double-underlined and bold amino acid
(A114) in Ent-1 corresponds to an amino acid insertion introducing an
irregularity in the heptad repeats. Mutations observed in M/P of
familial Mediterranean fever patients are given below the
sequence comprised between amino acids 667 and 727 (32, 33).
helix: whereas position
a displays only leucine, subsequent positions by rotating
around the helix were successively basic (KHKKHK ... at position
e, RQKRQK ... at position b), acidic
(EDEEDE ... at position f), neutral (KTEKTE ... at
position c, TQQTQQ ... at position g), and
again predominantly acidic (DKEDEEDEE ... at position d). In contrast, Ent-1 did not display the same geometrical
regularity, and some of the heptad repeats contained a basic amino acid
instead of a leucine or aromatic residue, as mentioned above (Fig.
2B).
View larger version (30K):
[in a new window]
Fig. 2.
Comparison of enterophilin heptad
repeats. End view of the Ent-2-S (A) and enterophilin-1
(B) leucine zipper, looking from the amino terminus. The
letters inside each circle represent standard nomenclature
for the seven amino acids found in unique positions in a coiled coil.
The sequence of the residues at each position of the helix is to be
read outward of the helix. For enterophilin-1, the asterisk
showed the irregularity in repeats due to an alanine insertion at
position 114.
View larger version (36K):
[in a new window]
Fig. 3.
Subcellular localization of
enterophilin-1. A, wild type and recombinant plasmids
(pCI and pCI-Ent-1, respectively) were used for in
vitro coupled transcription/translation in the presence of
[35S]methionine and a reticulocyte lysate (Promega).
B, COS-7 cells were grown in 60-mm dishes, transfected with
wild type (pCI) or recombinant vector (pCI-Ent-1), and analyzed 48 h after transfection by Western blotting using the anti-peptide
antibody. C, Weiser fraction 6 homogenate (H) was
fractionated into nuclei (N) and postnuclear supernatant
(S) as described under "Experimental Procedures." 100 µg of protein from each fraction were analyzed by Western blotting
using the antibody directed against the carboxyl-terminal peptide of
leucine zipper (top panel). Villin and lamin B were detected
using specific monoclonal antibodies (middle and
bottom panels, respectively). Note that besides the typical
67-kDa lamin B form, an additional 70-kDa band might represent the
lamin B2 isotype expressed at lower levels in mammals. D,
COS-7 cells were transfected with pGFP-Ent-1 (D,
1 and 2) or with wild type pGFP-C1 (D,
3), cultured for 65 h, and analyzed by fluorescence
microscopy (D, 1 and 3) or observed by
light microscopy (D, 2).
View larger version (52K):
[in a new window]
Fig. 4.
Northern blot analysis of enterophilin-1
mRNA in various guinea pig tissues. Total RNA isolated from
various guinea pig tissues (30 µg) was run on 1% agarose, and
ethidium bromide staining of rRNA is presented (bottom
panel). After migration, RNA was transferred onto a nylon membrane
and hybridized with 32P-labeled 850-bp enterophilin-1 probe
(top panel). After autoradiography, the nylon membrane was
stripped and rehybridized with the glyceraldehyde-3-phosphate
dehydrogenase probe as a loading control (middle
panel).
View larger version (39K):
[in a new window]
Fig. 5.
Expression of enterophilin-1 along the small
intestine horizontal and vertical differentiation axes.
A, 100 µg of protein from homogenates corresponding to
different guinea pig intestinal segments from duodenum to colon were
analyzed by Western blotting with anti-peptide antibody. P,
proximal; M, middle; D, distal. For the colon,
the distal segment was divided into D1 and D2 (more distal than D1).
B, enterocytes were separated into nine different fractions
along the villus-crypt axis according to Weiser (42) and homogenized.
100 µg of protein from homogenates were analyzed by Western blotting
with anti-peptide antibody.
View larger version (31K):
[in a new window]
Fig. 6.
Expression of enterophilin-1-related protein
during Caco-2 cell spontaneous growth and differentiation.
A, cells grown in 6 well plates were harvested and counted
with a Coulter cell counter at the indicated times. The values are the
means ± S.E. of triplicate determinations. B, 50 µg
of total cellular protein were separated by 7% SDS-polyacrylamide gel
electrophoresis, and expression of enterophilin-1-related protein was
followed with the anti-peptide antibody. C, sucrase
(SI) and alkaline phosphatase (AP) activities
were determined at the indicated times as described under
"Experimental Procedures." Data (means of duplicate determinations)
are compared with densitometric analysis of enterophilin-1-related
protein expression (Ent 1).
View larger version (24K):
[in a new window]
Fig. 7.
Expression of enterophilin-1-related protein
during butyrate-induced Caco-2 cell differentiation. Caco-2 cells
were treated with 5 mM butyrate or left untreated. At the
indicated times, expression of enterophilin-1-related protein was
analyzed by Western blotting with the anti-peptide antibody
(A) and quantified by densitometry (B) in the
absence ( ) and presence (
) of butyrate. Alkaline phosphatase
(C) and sucrase-isomaltase (D) activities were
determined in the cells in the absence (
) and presence (
and
) of butyrate. The values are the means of duplicate
experiments.
View larger version (18K):
[in a new window]
Fig. 8.
Effect of enterophilin-1 overexpression on
HT-29 cell growth and differentiation. HT-29 cells were
transiently transfected as described under "Experimental
Procedures" with empty vector pcDNA3 (white
box) or with pcDNA3-Ent-1 (shaded box).
At the indicated times, they were analyzed for
[3H]thymidine incorporation (A) or harvested
with trypsin/EDTA and counted (B). In parallel, cells were
scraped in PBS, and alkaline phosphatase specific activity was
measured. Data are expressed as increases in enterophilin-1-transfected
cells compared with cells treated with empty vector (C). All
the values are the means ± S.E. from three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix, which contributes to the
interface between dimerized helices (60, 61). It thus remains to be
determined whether all enterophilins display any ability to
dimerize by discriminating between zipper and non-zipper structures
(62). Further investigations will be undertaken to characterize the
other members of the enterophilin family. Accordingly, it is
interesting to note that multiple transcripts have already been
reported for other members of the B30.2 family such as Ro/SS-A, RoRet,
or MID1 (30, 63-65). For the latter gene, other mutations responsible
for Opitz syndrome were recently described, and authors reported the
presence of at least six isoforms of mRNA (66).
helix,
as discussed above. No classical nuclear localization signal was
apparent in the protein (68). Alternative mechanisms such as transport
by a cofactor as described for human MxB protein (69) may account for
its nuclear localization. Furthermore, the presence of several putative
phosphorylation sites in the sequence of enterophilin-1 suggests a
role of phosphorylation events in its intracellular localization.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Henry and P. Pontarotti for fruitful discussions, Drs. J. P. Salles and F. Gaits for critical reading of the manuscript, and Dr. R. Eyssen-Hernandez for help with graphics. We gratefully acknowledge the contribution of L. Vrancken in collecting literature information. We also thank Y. Jonquière for reading the English manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from the Association pour la Recherche contre le Cancer and from the Ligue Régionale contre le Cancer de Midi-Pyrénées.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) AF 126833 (Ent-1), AF 126831 (Ent-2-L), and AF 126832 (Ent-2-S).
To whom correspondence should be addressed: INSERM Unité
326, Hôpital Purpan, 31059 Toulouse Cedex, France. Tel.:
33-5-61-77-94-00; Fax: 33-5-61-77-94-01; E-mail:
Ama.Gassama@purpan.inserm.fr.
§ A recipient of a fellowship from the Association pour la Recherche contre le Cancer.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M009784200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bp, base pair(s); kb, kilobase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Ent-1, enterophilin-1.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 537-562[Medline] [Order article via Infotrieve] |
2. | Loeffler, M., Birke, A., Winton, D., and Potten, C. (1993) J. Theor. Biol. 160, 471-491[CrossRef][Medline] [Order article via Infotrieve] |
3. | Loeffler, M., Bratke, T., Paulus, U., Li, Y. Q., and Potten, C. S. (1997) J. Theor. Biol. 186, 41-54[CrossRef][Medline] [Order article via Infotrieve] |
4. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 461-480[Medline] [Order article via Infotrieve] |
5. | Cheng, H., and Leblond, C. P. (1974) Am. J. Anat. 141, 503-520[Medline] [Order article via Infotrieve] |
6. | Cheng, H. (1974) Am. J. Anat. 141, 521-536[Medline] [Order article via Infotrieve] |
7. | Gordon, J. I. (1989) J. Cell Biol. 108, 1187-1194[Medline] [Order article via Infotrieve] |
8. | Schmidt, G. H., Wilkinson, M. M., and Ponder, B. A. J. (1985) Cell 40, 425-429[Medline] [Order article via Infotrieve] |
9. | Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P. J., and Clevers, H. (1998) Nat. Genet. 19, 379-383[CrossRef][Medline] [Order article via Infotrieve] |
10. | Suh, E., and Traber, P. G. (1996) Mol. Cell. Biol. 16, 619-625[Abstract] |
11. |
Lorentz, O.,
Duluc, I.,
De Arcangelis, A.,
Simon-Assmann, P.,
Kedinger, M.,
and Freund, J. N.
(1997)
J. Cell Biol.
139,
1553-1565 |
12. |
Lynch, J.,
Suh, E.-R.,
Silberg, D. G.,
Rulyack, S.,
Blanchard, N.,
and Traber, P. G.
(2000)
J. Biol. Chem.
275,
4499-4506 |
13. | Louvard, D., Kedinger, M., and Hauri, H. P. (1992) Annu. Rev. Cell Biol. 8, 157-195[CrossRef] |
14. | Semenza, G. (1986) Annu. Rev. Cell Biol. 2, 255-313[CrossRef] |
15. | van Beers, E. H., Büller, H. A., Grand, R. J., Einerhand, A. W. C., and Dekker, J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 197-262[Abstract] |
16. |
Boll, W.,
Schmid-Chanda, T.,
Semenza, G.,
and Mantei, F.
(1993)
J. Biol. Chem.
268,
12901-12911 |
17. |
Takemori, H.,
Zolotaryov, F. N.,
Ting, L.,
Urbain, T.,
Komatsubara, T.,
Hatano, O.,
Okamoto, M.,
and Tojo, H.
(1998)
J. Biol. Chem.
273,
2222-2231 |
18. | Delagebeaudeuf, C., Gassama, A., Collet, X., Nauze, M., and Chap, H. (1996) Biochim. Biophys. Acta 1303, 119-126[Medline] [Order article via Infotrieve] |
19. |
Delagebeaudeuf, C.,
Gassama-Diagne, A.,
Nauze, M.,
Ragab, A.,
Li, R. Y.,
Capdevielle, J.,
Ferrara, P.,
Fauvel, J.,
and Chap, H.
(1998)
J. Biol. Chem.
273,
13407-13414 |
20. | Traber, P. G., and Silberg, D. G. (1996) Annu. Rev. Physiol. 58, 275-297[CrossRef][Medline] [Order article via Infotrieve] |
21. | Taylor, J. K., Boll, W., Levy, T., Suh, E., Siang, S., Mantei, N., and Traber, P. G. (1997) DNA Cell Biol. 16, 1419-1428[Medline] [Order article via Infotrieve] |
22. | Vernet, C., Boretto, J., Mattéi, G., Takahashi, M., Jack, L. J. W., Mather, I. H., Rouquier, S., and Pontarotti, P. (1993) J. Mol. Evol. 37, 600-612[Medline] [Order article via Infotrieve] |
23. | Seto, M. H., Liu, H. L., Zajchowski, D. A., and Whitlow, M. (1999) Proteins 35, 235-249[CrossRef][Medline] [Order article via Infotrieve] |
24. | Henry, J., Ribouchon, M.-T., Depetris, D., Matteï, M.-G., Offer, C., Tazi-Ahnini, R., and Pontarotti, P. (1997) Immunogenetics 46, 383-395[CrossRef][Medline] [Order article via Infotrieve] |
25. | Henry, J., Ribouchon, M.-T., Offer, C., and Pontarotti, P. (1997) Biochem. Biophys. Res. Commun. 235, 162-165[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Henry, J.,
Mather, I. H.,
McDermott, M. F.,
and Pontarotti, P.
(1998)
Mol. Biol. Evol.
15,
1696-1705 |
27. | Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856[Medline] [Order article via Infotrieve] |
28. | Iwata, Y., Nakayama, A., Murakami, H., Iida, K. I., Iwashita, T., Asai, N., and Takahashi, M. (1999) Biochem. Biophys. Res. Commun. 261, 381-384[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Cao, T.,
Borden, K. L.,
Freemont, P. S.,
and Etkin, L. D. J.
(1997)
J. Cell Sci.
110,
1563-1571 |
30. | Chan, E. K. L., Di Donato, F., Hamel, J. C., Tseng, C.-E., and Buyon, J. P. (1995) J. Exp. Med. 182, 983-992[Abstract] |
31. |
El-Hodiri, H. M.,
Che, S.,
Nelman-Gonzalez, M.,
Kuang, J.,
and Etkin, L. D.
(1997)
J. Biol. Chem.
272,
20463-20470 |
32. | The French Familial Mediterranean Fever Consortium. (1997) Nat. Genet. 17, 25-31[Medline] [Order article via Infotrieve] |
33. | The International Familial Mediterranean Fever Consortium. (1997) Cell 90, 797-807[Medline] [Order article via Infotrieve] |
34. | Quaderi, N. A., Schweiger, S., Gaudenz, K., Franco, B., Rugarli, E. I., Berger, W., Feldman, G. J., Volta, M., Andolfi, G., Gilgenkrantz, S., Marion, R. W., Hennekam, R. C. M., Opitz, J. M., Muenke, M., Ropers, H. H., and Ballabio, A. (1997) Nat. Genet. 17, 285-291[Medline] [Order article via Infotrieve] |
35. |
Cainarca, S.,
Messali, S.,
Ballabio, A.,
and Meroni, G.
(1999)
Hum. Mol. Genet.
8,
1387-1396 |
36. | Perry, J., Short, K. M., Romer, J. T., Swift, S., Cox, T. C., and Ashworth, A. (1999) Genomics 62, 385-394[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Jack, L. J. W.,
and Mather, I. H.
(1990)
J. Biol. Chem.
265,
14481-14486 |
38. | Ishii, T., Aoki, N., Noda, A., Achadi, T., Nakamura, R., and Matsuda, T. (1995) Biochim. Biophys. Acta 1245, 285-292[Medline] [Order article via Infotrieve] |
39. | Taylor, M. R., Peterson, J. A., Ceriani, R. L., and Couto, J. R. (1996) Biochim. Biophys. Acta 1306, 1-4[Medline] [Order article via Infotrieve] |
40. | Ye, T. Z., Gordon, C. T., Lai, Y. H., Fujiwara, Y., Peters, L. L., Perkins, A. C., and Chui, D. H. (2000) Gene (Amst.) 242, 337-345[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Ghadessy, F. J.,
Chen, D.,
Kini, M.,
Chung, M. C. M.,
Jeyaseelan, K.,
Khoo, H. E.,
and Yuen, R.
(1996)
J. Biol. Chem.
271,
25575-25581 |
42. |
Weiser, M. M.
(1973)
J. Biol. Chem.
248,
2536-2541 |
43. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual (Maniatis, T., ed), 2nd Ed. , pp. 7.1-7.53, Cold Spring Harbor Press, Cold Spring Harbor, NY. |
44. | Arcari, P., Martinelli, R., and Salvatore, F. (1984) Nucleic Acids Res. 12, 9179-9189[Abstract] |
45. | Raynal, P., Van Bergen en Henegouwen, P., Hullin, F., Ragab-Thomas, J. M. F., Fauvel, J., Verkleij, A., and Chap, H. (1992) Biochem. Biophys. Res. Commun. 186, 432-439[Medline] [Order article via Infotrieve] |
46. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
47. | Stott, D. (1991) in Methods in Molecular Biology (Murray, E. J., ed), Vol. 7 , pp. 327-335, Human Press Inc., Clifton, NJ |
48. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
49. | Burnette, W. N. (1981) Anal. Biochem. 112, 195-203[Medline] [Order article via Infotrieve] |
50. | Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve] |
51. | Rousset, M. (1986) Biochimie (Paris) 68, 1035-1040[Medline] [Order article via Infotrieve] |
52. | Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix Simon-Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. (1983) Biol. Cell 47, 323-330 |
53. |
Ding, Q.-M.,
Ko, T. C.,
and Evers, B. M.
(1998)
Am. J. Physiol.
275,
C1193-C1200 |
54. | Burbelo, P. D., Gabriel, G. C., Kibbey, M. C., Yamada, Y., Kleinman, H. K., and Weeks, B. S. (1994) Gene (Amst) 139, 241-245[CrossRef][Medline] [Order article via Infotrieve] |
55. | Lu, R., Yang, P., O'Hare, P., and Misra, V. (1997) Mol. Cell. Biol. 17, 5117-5126[Abstract] |
56. |
Marini, M. G.,
Chan, K.,
Casula, L.,
Kan, Y. W.,
Cao, A.,
and Moi, P.
(1997)
J. Biol. Chem.
272,
16490-16497 |
57. | Rook, F., Weisbeek, P., and Smeekens, S. (1998) Plant Mol. Biol. 37, 171-178[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Bikle, D. D.,
Munson, S.,
Morrison, N.,
and Eisman, J.
(1993)
J. Biol. Chem.
268,
620-626 |
59. |
Bikle, D. D.,
Munson, S.,
and Komuves, L.
(1996)
J. Biol. Chem.
271,
9075-9083 |
60. | Krylov, D., Mikhaleinko, I., and Vinson, C. (1994) EMBO J. 13, 2849-2861[Abstract] |
61. |
Junius, F. K.,
O'Donoghue, S. I.,
Nilges, M.,
Weiss, A. S.,
and King, G. F.
(1996)
J. Biol. Chem.
271,
13663-13667 |
62. | Hirst, J. D., Vieth, M., Skolnicl, J., and Brooks, C. L., III. (1996) Protein Eng. 8, 657-662 |
63. | Itoh, K., Itoh, Y., and Frank, M. B. (1991) J. Clin. Invest. 87, 177-186[Medline] [Order article via Infotrieve] |
64. |
Ruddy, D. A.,
Kronmal, G. S.,
Lee, V. K.,
Mintier, G. A.,
Quintana, L.,
Domingo, R., Jr.,
Meyer, N. C.,
Irrinki, A.,
McClelland, E. E.,
Fullan, A.,
Mapa, F. A.,
Moore, T.,
Thomas, W.,
Loeb, D. B.,
Harmon, C.,
Tsuchihashi, Z.,
Wolff, R. K.,
Schatzman, R. C.,
and Feder, J. N.
(1997)
Genome Res.
7,
441-456 |
65. |
Buchner, G.,
Montini, E.,
Andolfi, G.,
Quaderi, N.,
Cainarca, S.,
Messali, S.,
Bassi, M. T.,
Bhallabio, A.,
Meroni, G.,
and Franco, B.
(1999)
Hum. Mol. Genet.
8,
1397-1407 |
66. |
Cox, T. C.,
Allen, L. R.,
Cox, L. L.,
Hopwood, B.,
Goodwin, B.,
Haan, E.,
and Suthers, G. K.
(2000)
Hum. Mol. Genet.
9,
2553-2562 |
67. |
Schultz, J.,
Milpetz, F.,
Peer, B.,
and Ponting, C. P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5857-5864 |
68. | Dingwall, C., and Laskey, R. A. (1991) Trends Biochem. Sci. 16, 478-481[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Melen, K.,
and Julkunen, I.
(1997)
J. Biol. Chem.
272,
32353-32359 |
70. |
Harada, H.,
Harada, Y.,
O'Brien, D. P.,
Rice, D. S.,
Naeve, C. W.,
and Downing, J. R.
(1999)
Mol. Cell. Biol.
19,
3808-3815 |
71. | Inoue, S., Urano, T., Ogawa, S., Saito, T., Orimo, A., Hosoi, T., Ouchi, Y., and Muramatsu, M. (1999) Biochem. Biophys. Res. Commun. 261, 412-418[CrossRef][Medline] [Order article via Infotrieve] |
72. |
Orimo, A.,
Inoue, S.,
Ikeda, K.,
Noji, S.,
and Muramatsu, M.
(1995)
J. Biol. Chem.
270,
24406-24413 |
73. |
Tidow, N.,
Chen, X.,
Müller, C.,
Kawano, S.,
Gombard, A. F.,
Fischel-Ghodsian, N.,
and Koeffler, P.
(2000)
Blood
95,
1451-1455 |
74. |
Chen, X.,
Bykhovskaya, Y.,
Tidow, N.,
Hamon, M.,
Bercovitz, Z.,
Spirina, O.,
and Fischel-Ghodslan, N.
(2000)
Proc. Soc. Exp. Biol. Med.
224,
32-40 |