From the
We have cloned a cDNA corresponding to a novel gene from a human
epithelial cell line by subtractive hybridization and polymerase chain
reaction techniques. This gene is expressed at the message level and at
the protein level in a lung alveolar type II-like epithelial cell line
but not in lung fibroblasts. In adult human tissues, the mRNA for this
gene was detected only in the heart and the skeletal muscle, but not in
the brain, placenta, whole lung, liver, or kidney. We have named this
gene I
The establishment of specialized cell types during development
involves commitment of multipotential stem cells to specific lineages
which is associated with expression of cell type-specific genes. It has
long been established that morphogenesis of epithelial tissues involves
an interaction with the adjacent mesenchyme during embryonic
development (reviewed in Ref. 1). Alveolar type II cells are
specialized epithelial cells serving important functions in lung
development and repair post-injury
(2) . To understand the
biology of epithelial cell function, we attempted to clone genes that
are expressed in type II-like epithelial cells, such as the lung
adenocarcinoma cell line A549, but not in lung mesenchymal cells.
We
report the cloning of the cDNA for a differentially expressed protein
that has significant similarity to I
NF-
I
The ankyrin-like repeat sequence
is a 33-amino acid motif, first described in the yeast cell cycle
control proteins, which consists of a TPLHLA core amino acid sequence
and additional well conserved residues
(12, 21) . The
number and the nature of the conserved amino acids can vary in
different proteins. A number of proteins containing ankyrin-like
repeats are involved in cell growth and differentiation
(12, 21) . It is becoming increasingly apparent that
ankyrin repeats mediate protein-protein interactions which are
essential in many biological functions, including cellular signaling
events
(22, 23, 24) .
The cDNA sequence of
I
Cell Culture All cell lines were obtained from American Type Culture Collection
(ATCC). The lung adenocarcinoma cell line A549 was maintained in
Dulbecco's modified Eagle's medium:F-12 (1:1) (Life
Technologies, Inc.) containing 10% fetal bovine serum (HyClone). The
lung fibroblast cells CCL-202 and HeLa cells were maintained in 10%
fetal bovine serum-supplemented Dulbecco's modified Eagle's
medium containing 1 or 4.5 mg/liter of glucose, respectively. F9 cells
were maintained as reported previously
(25) . Subtraction, PCR, and Northern Analyses
The fusion protein used in
electrophoretic mobility shift assays was purified from inclusion
bodies as follows. The insoluble pellet was solubilized in 8 M
urea and subsequently dialyzed against 50 mM Tris-HCl, pH 7.5.
The renatured protein was purified by affinity chromatography on
glutathione-agarose. The protein was eluted by continuous slow stirring
of the beads at 4 °C in a buffer containing 50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM reduced
glutathione, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride. The eluted protein was dialyzed against
50 mM Tris-HCl, pH 7.5. The purified protein was divided into
aliquots and stored at -80 °C. The protein was 98% pure as
revealed by SDS-PAGE. The soluble GST control protein was treated with
8 M urea and subjected to similar renaturation steps. The
identity of the GST protein and the GSTI
The
DNA sequences of the PCR products were translated in all three reading
frames and compared with available sequences in the gene bank. The DNA
sequences of all of the PCR-amplified fragments were bound by that of
the PCR primers showing primer-specific amplification of cDNAs. The
sequences of some of the clones were derived from known receptor
tyrosine kinases. The sequence of one clone revealed the presence of an
ankyrin-like repeat motif. Since this structural motif is often present
in proteins involved in cell cycle control and tissue differentiation,
we were persuaded to further characterize this clone. Preliminary
analyses indicated that the mRNA specific for this PCR fragment was
also expressed in HeLa cells, a cervical carcinoma cell line of
epithelial origin. We therefore proceeded to isolate the full-length
cDNA from a HeLa cell-derived cDNA library.
For sequencing, the DNA inserts
from the
We report the cloning and characterization of the cDNA for a
molecule which is differentially expressed in epithelial cell types but
not in fibroblasts and is related to the I
Among the presently characterized I
The ankyrin repeats
typically contain a core amino acid sequence (TPLH) and sequences
outside of this core are extremely variable. Different repeats within a
given I
In keeping with this argument, our data
indicate that I
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Gary Nabel for providing us with plasmid
constructs containing the p65 cDNA and Sankar Ghosh for the p50
expression vector and pBIIXLUC. We thank Lynna Stone-Infeld for
assistance with data base searches and Mandy Abkowicz for technical
assistance. We thank members of the Pulmonary and Critical Care Section
for numerous helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
BR (for I
B-related) since its 52-kDa protein product
has significant homology to the I
B family of proteins which
function as inhibitory cytoplasmic retention proteins for the
vertebrate rel/NF-
B transcription factors. Although the important
role of NF-
B in gene activation in cells of the immune system is
now well established, a similar role in other cell types or in
vertebrate development is less clear. The deduced amino acid sequence
of I
BR has the most significant homology to the Drosophila protein Cactus which inhibits the function of the NF-
B-like
protein Dorsal. In electrophoretic mobility shift experiments, I
BR
inhibited the ability of the p50:p65 NF-
B heterodimer to bind DNA.
The DNA binding ability of the p50 homodimer but not the p65 homodimer
was drastically inhibited by I
BR. In transfection experiments,
overexpression of I
BR significantly inhibited NF-
B-dependent
transcription from the Ig
enhancer. This new member of the I
B
family of proteins, I
BR, may play an important role in regulation
of NF-
B function in epithelial cells.
B proteins. Therefore, we have
named this protein I
BR (for I
B-related). Among the I
B
proteins, overall, Cactus was most similar to I
BR. Cactus inhibits
the transcription activation function of the NF-
B-like protein
Dorsal in Drosophila, analogous to the NF-
B-I
B
interaction in mammalian cells
(3, 4) . Dissociation of
Cactus from Dorsal results in translocation of Dorsal to the nucleus
where it activates genes involved in formation of the dorsoventral axis
(5, 6) .
B was initially characterized as a
heterodimer of a 50-kDa protein (p50) and a 65-kDa protein (p65, now
called RelA) that was retained in the cytoplasm by a protein called
I
B
(7, 8, 9) . Activation of NF-
B,
induced by various agents, including mitogens and cytokines, results in
its dissociation from I
B, thereby allowing its translocation to
the nucleus to activate genes involved in immune functions
(10, 11, 12, 13) . It appears that,
unlike what was previously thought, phosphorylation of I
B
may
trigger its degradation by cytoplasmic proteases while still complexed
to NF-
B, rather than cause it to dissociate from NF-
B
(14) .
B proteins whose cDNA sequences are known
include I
B
and its avian homolog pp40
(15, 16) , Bcl-3
(17) , the product of the
bcl-3 oncogene
(18) and I
B
, a
lymphoid-specific form of I
B
(19, 20) . The
sequence of the I
B
gene is presently unavailable. I
B
proteins typically contain ankyrin-like repeats, short acidic domains,
and consensus sequences for phosphorylation by protein kinase A,
protein kinase C, and casein kinase II
(7, 8, 9) .
BR has an open reading frame of 481 amino acids with three
ankyrin-like repeats in its carboxyl terminus. By Western blotting
techniques, we identified a 52-kDa protein in epithelial cell lines,
but not in fibroblasts using antisera raised against a bacterially
expressed fusion protein (glutathione S-transferase linked to
the 52-kDa protein). We tested if the result of the data base search
showing similarity of the I
BR protein with multiple I
B-like
proteins held up in functional assays. All I
B proteins inhibit the
binding of specific NF-
B protein(s), as demonstrated by
electrophoretic mobility shift assays. I
BR inhibited the DNA
binding ability of an NF-
B complex present in nuclear extracts
prepared from interleukin-1 (IL-1)
(
)
-activated
HeLa cells. The binding of a p50 homodimer but not a p65 homodimer was
also inhibited by I
BR. In transfection experiments, I
BR
significantly inhibited NF-
B-dependent transcription from the
Ig
enhancer. We postulate that I
BR may play an important role
in the regulation of NF-
B function in epithelial cells.
Synthesis of cDNA and biotinylated Poly(A)
Poly(A) RNA
RNA from A549 cells was used
in first strand cDNA synthesis (using random hexamers), whereas that
from CCL-202 cells was used for preparation of biotinylated RNA as
described by Forster et al.(26) .
Enrichment of A549-specific mRNA
To remove the
mRNA species common between A549 cells and CCL-202 cells, a 30-fold
molar excess of biotinylated mRNA (from CCL-202 cells) over cDNA (from
A549 cells) was used in the solution hybridization reaction containing
0.5 M NaCl which was carried out for 24 h at 68 °C. This
resulted in a Ro T of 67.5 (mg of RNA/ml of
hybridization mix) over 1000 per day which is sufficient for
hybridization of even less abundant classes of cellular mRNA sequences
which are present in both cell types
(26) . cDNA:biotinylated
mRNA hybrids were removed with streptavidin followed by phenol
extraction and the residual cDNA was recovered by ethanol precipitation
and was used as template for PCR amplification. The enrichment step was
repeated twice.
PCR Amplification, Cloning, and Sequencing of
PCR-amplified Fragment
Two regions in the catalytic domain of
type III receptor tyrosine kinases were used to derive oligonucleotide
primers for PCR amplification. The sequence of the 5` oligonucleotide
primer was 5`-GTCGACAAC/TCTGTTGGGA/GGCCTGC- 3` while that of the 3`
primer was 5`-GAATTCAGCAGCCAGGTCTCT/GGTG-3`; the underlined sequences
indicate SalI and EcoRI restriction enzyme sites in
the 5` and 3` oligonucleotide, respectively. After PCR amplification,
the amplified DNA was digested with SalI and EcoRI
and cloned into the bacteriophage vector M13mp18. Several recombinant
clones were analyzed by sequencing by the dideoxy method
(27) .
Northern Analyses
For Northern analyses, 10-µg
aliquots of total cellular RNA were denatured with formamide and
formaldehyde and then loaded onto 1% agarose gels. The RNA was
transferred onto nylon membranes (Micron Separations Inc., Westboro,
MA), and hybridization was performed with P-labeled probes
following procedures recommended by the manufacturer. Screening of an Epithelial Cell-specific cDNA Library and DNA Sequencing
For isolation of the full-length cDNA, a HeLa cell cDNA library in
gt11 was screened with the PCR-amplified fragment following
standard procedures. The cDNA inserts were subcloned into M13 phage
vectors mp18 and mp19 for making deletion constructs and DNA
sequencing. A series of nested deletions of the cloned fragments were
carried out in an unidirectional (5`-3`) manner using the Cyclone I
Biosystem (International Biotechnologies, Inc.). DNA sequencing was
performed using the Sequenase kit (U. S. Biochemical Corp.). Both
strands were sequenced to obtain an accurate sequence. A composite
sequence was derived from overlapping sequences of the progressively
deleted fragments. Comparisons with data base sequences were performed
using the Blastmail program of the National Center of Biotechnology
Information. Construction of a Glutathione S-Transferase-I
BR (GSTI
BR)
Fusion Protein and Preparation of Antiserum The pGEX-2T vector (Pharmacia Biotech Inc.) was used for bacterial
expression of an in-frame GSTI
BR fusion protein
(28) . The
recombinant vector was transformed into bacteria deficient in lon
protease (strain NB42 of Escherichia coli). Unless otherwise
indicated, bacteria were grown at 30 °C in all subsequent steps. A
large fraction of the bacterially expressed fusion protein was trapped
in inclusion bodies which were recovered following manufacturer's
recommendations. For immunization of rabbits, the protein was purified
by SDS-PAGE and electroelution.
BR fusion protein was
confirmed by Western blotting using anti-GST antibody. Western Blot Analyses Western blotting was performed by electrophoretic transfer of proteins
from SDS-polyacylamide gels onto Immobilon-P (Millipore) membranes.
Following incubation of the membrane with primary antibody, the
membrane was further processed with alkaline-phosphatase-conjugated
mouse anti-rabbit antibody and the detection system of Vector
Laboratories. Electrophoretic Mobility Shift Assays Nuclear extracts were prepared from uninduced and IL-1-treated HeLa
cells as reported previously
(29) . Nuclear extracts or
individual proteins were preincubated in the presence or absence of
antibodies or competitor proteins for 30 min on ice in a buffer
containing 10 mM Hepes, pH 7.9, 50 mM NaCl, 1
mM dithiothreitol, 1 mM EDTA and 10% glycerol, 1
mM phenylmethylsulfonyl fluoride, 2.5 µg/ml
poly(dI)
poly(dC), and 200 µg/ml bovine serum albumin before
addition of the labeled probe and further incubation at room
temperature for 15 min. A
P-labeled oligonucleotide
containing the NF-
B sequence from the IL-6 promoter was used as
the probe. The binding reactions were analyzed by electrophoresis on 6%
native polyacrylamide gels (acrylamide:bisacrylamide = 30:1).
Electrophoresis was carried out at 200 V in 0.5
TBE (1
= 0.05 M Tris base, 0.05 M boric acid, and 1.0
mM EDTA) at 4 °C. Gels were dried and subjected to
autoradiography. Transfections Transfections in F9 cells were carried out as reported previously
(25) . Luciferase assays were performed using the Luciferase
Assay System of Promega (Wisconsin) and the luciferase activity was
measured in the LB 9501 luminometer of Berthold, Inc. (Bad Wildbad,
Federal Republic of Germany).
Subtraction Hybridization and PCR
An enriched
population of A549 cell-specific cDNA was obtained by subtractive
hybridization with mRNA from lung fibroblast cells. This cDNA was
subjected to PCR using degenerate primers from conserved domains
present in type III receptor tyrosine kinases which mediate the
proliferation and differentiation of many cells by a variety of growth
factors
(30) . Analysis of the PCR amplification product of
A549-specific cDNA by agarose gel electrophoresis revealed amplified
DNA fragments in the range from 200 to 600 bp (data not shown).
cDNA Isolation and Characterization
A HeLa cell
cDNA library in the phage vector gt11 was screened with the 276-bp
PCR fragment for isolation of the full-length cDNA. This resulted in
the isolation of three independent clones. Southern blot analysis at
high stringencies showed the presence of a
2-kb insert in one of
these clones, a 0.5-kb fragment of which showed specific hybridization
to the probe. The other two independent clones contained sequences
overlapping with the 2-kb insert.
phage clones were subcloned in both orientations into
M13 phage vectors. Sequential deletions of the inserts were obtained
for complete sequence information and a composite sequence was derived
from the overlapping sequences. The cDNA was found to contain an open
reading frame of 481 amino acids (Fig. 1); there are also 324 bp
of 5`-untranslated region upstream (data not shown). The ATG codon at
nucleotide position 325 is the putative start codon for the full-length
gene for the following reasons: ( a) this ATG codon is in the
context of a Kozak sequence
(31) and the longest open reading
frame and ( b) there are stop codons in all reading frames 5`
of this ATG codon. Based on the results of the Northern analyses, which
showed a
5.5-kb message hybridizing to the probe (Fig. 3),
we predict that we are missing 3.5 kb of the 3`-untranslated sequence.
Examples of other genes whose transcripts have been shown to contain
long untranslated 3`-ends are the vinculin gene
(32) and the
estrogen receptor gene
(33) .
Figure 1:
Nucleotide and predicted amino acid
sequence of the coding region of the cDNA encoding IBR. The
potential protein kinase A, protein kinase C, and casein kinase II
phosphorylation sites are indicated by the overlines, the
underlines, and the broken lines, respectively. The
boxed sequences contain the three ankyrin-like repeat motifs.
The termination codon is denoted by an
asterisk.
Figure 3:
Differential expression of IBR mRNA
and protein in epithelial cell lines. A, mRNA expression.
Total cytoplasmic RNA was isolated from human tracheal epithelial cell
line ( HTE), human alveolar type II-like epithelial cell line
( A549), and a human lung fibroblast cell line
( CCL-202). The RNA (20 µg/lane) was subjected to Northern
blot analysis by using the
P-labeled PCR fragment (276 bp)
as the hybridization probe. RNA molecular size standards are shown on
the left in kilobases. Ethidium bromide staining of the same
RNAs loaded in a duplicate gel confirmed similar loading of RNA.
B, protein expression. HeLa cells or CCL-202 cells were lysed
by boiling in SDS-polyacrylamide gel sample loading buffer and were
subjected to SDS-PAGE. The fractionated proteins were transferred onto
nitrocellulose membrane by electroblotting. Strips of nitrocellulose
containing bound protein were incubated with preimmune serum or
antiserum from a rabbit immunized with the GST fusion protein.
Antibodies bound to proteins were detected by subsequent incubation
with alkaline phosphatase-conjugated goat anti-rabbit IgG followed by
alkaline phosphatase substrate solution. The positions of protein size
markers are indicated on the left. The bottom arrow denotes the expected I
BR band. The upper arrow denotes a cross-reacting species of greater molecular
size.
The deduced amino acid sequence
of the cDNA was compared with available amino acid sequences of other
proteins in protein data bases using the BLAST e-mail server. BLAST
utilizes an alignment algorithm that identifies similarities without
need to introduce gaps and is therefore more stringent
(34) .
Data base searches revealed that the cDNA we isolated encoded a novel
sequence with the following characteristics: ( a) the amino
acid sequence between residues 363 and 471 in IBR containing the
three ankyrin repeats (Fig. 1) was similar to ankyrin repeats
present in I
B-like proteins. The overall similarities were as
follows: (i) amino acids 363-429 (containing two repeats) and
437-471 (comprising the third repeat) in I
BR showed a 32%
identity, 49% similarity and a 28% identity, 51% similarity to repeats
II-III and repeat IV, respectively, in the Drosophila I
B-like protein Cactus
(4) ; (ii) amino acids
363-471 in I
BR showed a 33% identity, 49% similarity with a
stretch containing repeats II, III, and IV present in the precursor of
the p50 subunit of NF-
B, p105
(12) , and in I
B
, a
lymphoid-specific form of I
B whose sequence is identical to the
C-terminal part of p105; and last (iv) amino acids 363-424 and
436-464 in I
BR showed a 40% identity, 54% similarity and a
34% identity, 58% similarity to repeats II-III and repeat V,
respectively, in pp40, the avian homolog of I
B
(15, 16) . The ankyrin repeats in I
BR also showed
similarity to those in the erythrocyte protein ankyrin
(22) and
the G9a protein of presently unknown function
(35) .
( b) A 36-amino acid stretch (residues 304-339) rich in
glutamate residues showed a 27% identity and 47% similarity with a
homologous stretch in the Cactus protein. This region was also found to
be similar to a glutamate-rich region in the G9a protein
(35) .
Fig. 2
illustrates the similarity of I
BR with the
Drosophila protein Cactus, which overall was most homologous
to I
BR. Protein motif searches showed the presence of consensus
sequences for protein kinase A, protein kinase C, and casein kinase II
phosphorylations in I
BR which are also commonly found in I
B
proteins (Fig. 1). It is important to note that no similarity was
detected with the Rel homology domain in the Rel family of
transcription factors (NF-
B), some of which contain ankyrin-like
repeats in their precursor forms. It therefore seemed unlikely that the
protein encoded by this cDNA was an NF-
B-like transcription
factor. Again, although the primers for the PCR amplification reaction
were derived from receptor tyrosine kinases, the amino acid sequence of
I
BR did not reveal characteristic motifs present in such
receptors. I
BR appears to have been a target in the amplification
reaction because of coincidental similarity of two regions in the gene,
corresponding to the ends of the 276-bp PCR fragment, to the DNA
sequences of the PCR primers. In summary, the overall similarity of the
I
BR protein was greatest with the I
B proteins than with other
ankyrin-repeat proteins such as members of the Notch family of proteins
(36) . Therefore, we named this protein I
BR for
I
B-related.
Figure 2:
Summary of the structural features of
IBR and sequence comparisons. A, a schematic
representation of the primary sequence of I
BR and the Cactus
protein. The stippled boxes denote the acidic domains in the
two proteins, whereas the cross-hatched boxes indicate the
ankyrin-like repeats. B, an alignment of the three
ankyrin-like repeats in I
BR with the general consensus sequence
for ankyrin repeats. (C). An alignment of the amino acid sequences of
( a) I
BR and Cactus across the ankyrin-like repeats and
( b) across the acidic amino acid-rich
regions.
Expression of I
It was expected that the
PCR-amplified fragment would be expressed in epithelial cells, but not
in fibroblasts, since the template for this reaction was a residue of
subtractive hybridization. We investigated by Northern blot analysis if
indeed this was true. As illustrated in Fig. 3 A,
IBR mRNA and Protein in Epithelial
Cells but Not in Fibroblasts
BR-specific message of
5.5 kb was detected in A549 cells and
in human tracheal epithelial cells. A much smaller transcript of
0.6 kb, insufficient in size to account for the full-length
I
BR protein, was also detected (this transcript ran off the
particular gel shown in Fig. 3 A but is shown in
Fig. 4
). No hybridization was detected with RNA derived from the
human lung fibroblast cell line CCL-202. Northern blot analyses
therefore indicated that I
BR transcripts are expressed in
epithelial cells (at least at much higher levels) in preference to
fibroblasts. The profile of I
BR mRNA expression in other cell
lines is presently unknown.
Figure 4:
Expression of IBR mRNA in adult human
tissues by Northern blot analysis. A human multi-tissue Northern blot
(Clontech) was hybridized to the full-length
P-labeled
I
BR cDNA probe. The positions of RNA size markers are shown on the
left.
To confirm that the IBR protein was
expressed in intact cells, polyclonal antibodies were raised against a
bacterial fusion protein consisting of glutathione
S-transferase fused to amino acids 39-481 of the
I
BR protein (GSTI
BR). Western blots were prepared from HeLa
cells (since these were used to isolate the cDNA) and fibroblast cell
lysates. On these blots, immune sera but not preimmune sera detected a
band of approximately 52 kDa in lysates prepared from HeLa cells
(Fig. 3 B). A fainter second band almost twice in size to
this band (approximately 100 kDa in size) was also detected which may
be (i) unreduced homodimers or heterodimers of the same protein
(mediated by the ankyrin-like repeats) or (ii) a cross-reacting
homologous protein. A similar pattern was also identified in proteins
recovered from A549 cells (data not shown).
Expression of I
We investigated the steady state level of IBR-specific mRNA in Adult Human
Tissues
BR-mRNA
expression in adult human tissues by Northern blot analysis
(Fig. 4). A 5.5-kb transcript, similar in size to that expressed
in epithelial cells, was detected only in the heart and the skeletal
muscle (Fig. 4). A shorter transcript was detected in all other
tissues included in the analysis except for the brain (Fig. 4).
This shorter message, insufficient in size to code for a 52-kDa
protein, was also detected in epithelial cells and in fibroblasts. The
shorter transcript could be an alternately spliced species of the
I
BR gene. Although this gene is expressed in the alveolar type
II-like epithelial cell line A549, one possible reason for not
detecting the longer transcript in the lung could be due to the fact
that type II epithelial cells constitute only 10% of the adult lung
alveolar epithelium. The significance of relatively high expression of
the 5.5-kb message in muscle tissue is not presently understood.
I
To elucidate the biological function of the IBR Inhibits DNA Binding Activity of NF-
B
Proteins
BR,
the ability of the GSTI
BR fusion protein to inhibit the DNA
binding activity of NF-
B proteins was tested in electrophoretic
mobility shift assays. In these experiments, the
P-labeled
oligonucleotide probe containing an NF-
B-site was derived from the
IL-6 promoter
(25, 37) , and the NF-
B protein
targets used were (i) the p50:p65 heterodimer present in HeLa cell
nuclear extracts, prepared from cells induced with IL-1, a known
activator of NF-
B
(25, 38, 39, 40) , (ii) the p50
homodimer (of the bacterially expressed protein), and (iii) the p65
homodimer (of in vitro translated protein). In electrophoretic
mobility shift assays, the DNA binding ability of the p50:p65
heterodimer was inhibited by the GSTI
BR fusion protein
(Fig. 5 A). The DNA binding activity of the p50 homodimer
was significantly inhibited by the fusion protein in a dose-dependent
manner (Fig. 5 B). Included in the same experiment,
identical concentrations of GSTI
BR minimally inhibited the DNA
binding activity of the p65 homodimer (Fig. 5 B). The GST
polypeptide alone did not affect the DNA binding activity of any of
these proteins, indicating that the inhibition was mediated by the
I
BR part of the fusion protein. The inhibition of DNA binding
activity of the p50 homodimer is reminiscent of that of the lymphoid
cell-specific I
B protein I
B
(19, 20) .
The effect of I
BR on the DNA binding ability of c-Rel has not been
determined.
Figure 5:
The effect of IBR on the binding of
NF-
B proteins to a target sequence ( A and B) and
suppression of p50/p65-mediated activation of the Ig
enhancer
in F9 cells ( C). A, nuclear
extracts from uninduced and IL-1-treated HeLa cells were incubated with
P-labeled oligonucleotide containing the NF-
B
sequence from the IL-6 promoter in the presence or absence of
antibodies to the different subunits of NF-
B and to the c-Fos
protein (as a negative control) or in the presence and absence of 25 or
12.5 ng of GSTI
BR or equimolar amounts of GST as competitors.
B, the DNA binding reaction was carried out with purified p50
protein (Promega) or in vitro translated p65 in the presence
or absence of antibodies to the proteins, as indicated or in the
presence of 25, 12.5, or 5 ng of GSTI
BR or equimolar amounts of
GST. All other conditions were as in A. The binding reactions
were analyzed by electrophoresis on 6% native polyacrylamide gels. Gels
were dried and subjected to autoradiography. C, effect of
I
BR on NF-
B-dependent transcription. F9 cells were
transfected with 5 µg of the reporter plasmid pBIIXLUC containing
two
B sites from the Ig
enhancer driving the expression of a
luciferase reporter gene together with equimolar concentrations (1
µg each) of expression plasmids for the p50 and the p65 subunits of
NF-
B, 2 µg of expression vector for I
BR expressing the
I
BR-mRNA in the sense ( S) or antisense ( AS)
orientation and 2 µg of RSV
gal (a constitutive expression
vector for
-galactosidase for monitoring transfection efficiency).
24 h after transfection, cells were harvested and assayed for
-galactosidase and luciferase activity. The absolute basal
luciferase activity was, on average, 3500 relative light units in these
experiments. This is an average of three independent experiments with
deviations of no more than 10% between
experiments.
Inhibition of NF-
We also examined the effect of
IB-mediated Activation of the
Ig
Enhancer by I
BR
BR on NF-
B-mediated activation of the Ig
enhancer in F9
(a murine embryonal carcinoma cell line) cells which lack endogenous
Rel-like activity
(41) . F9 cells were transiently transfected
with a plasmid construct that contained two
B sites from the
Ig
enhancer linked to a luciferase reporter gene together with
expression vectors for the p50 and the p65 subunits of NF-
B. A
combination of p50 and p65 efficiently activated expression from the
B sites in the reporter construct (Fig. 5 C). When
the p50 and p65 expression vectors were cotransfected with an
expression vector for I
BR, expression of the reporter gene was
significantly inhibited (Fig. 5 C). However, no such
suppression was observed when a plasmid construct expressing antisense
( AS) I
BR was cotransfected with the NF-
B subunits
(Fig. 5 C). The expression of the I
BR expression
vector containing the I
BR cDNA in either orientation (sense or
antisense) by themselves had no effect on expression of the reporter
gene in the absence of the NF-
B subunits (Fig. 5 C).
Based on all the experiments in Fig. 5, it is too early to decide
which one of the mechanisms, inhibition of DNA binding activity of
NF-
B or inhibition of nuclear translocation of NF-
B or both,
are relevant to I
BR activity in vivo. It is also not
known if the in vivo target of I
BR is p50. However, the
data illustrated in Fig. 5indicate that I
BR has
I
B-like functions.
B family of proteins.
The 5.5-kb mRNA corresponding to this newly identified gene encodes a
protein product of 481 amino acids with a predicted molecular mass of
52 kDa. Intracellular expression of a protein of expected size was
confirmed using antisera raised against a GSTI
BR fusion protein.
B proteins, overall, the
amino acid sequence of the Drosophila Cactus protein was most
similar to that of I
BR. It is intriguing that while most I
B
proteins, including Cactus, contain five to seven ankyrin repeats, this
newly identified member of the family contains only three repeats.
Another recently identified gene ikbl (for I kappa B-like)
encodes a protein product containing two and a half ankyrin repeats,
which bear marked similarity to those present in the I
B family of
proteins
(42) . The difference in the repeat numbers between
I
BR and the other I
B proteins may reflect differences in the
nature of the interacting partners of the two classes of proteins. This
may have implications in the normal physiological function of I
BR.
It might be expected that the I
B proteins with fewer repeats such
as I
BR and Ikbl interact with a different set of NF-
B/Rel
proteins which respond to distinct stimuli.
B molecule are more variable than individual repeats
between members of the same class. In ankyrin, the different repeats
have different binding activities
(43) . It has been
hypothesized that the repeats in their folded conformation expose
variable residues that are capable of interacting with distinct protein
partners. By this line of reasoning, the fact that the ankyrin repeats
in I
BR showed the highest homology to those in Cactus and other
members of the I
B family suggested that I
BR might have
I
B-like functions.
BR can inhibit the DNA binding ability of
NF-
B. This is similar to the properties of most I
B proteins
which upon interaction with a Rel complex inhibit the DNA binding
ability of the Rel complex. Transfection experiments clearly indicate
that I
BR can inhibit activation of NF-
B sites mediated by a
combination of p50 and p65, as might be expected for I
B proteins.
However, the precise mechanism of this inhibition is unknown. The
inhibition could potentially be due to retention of the p50:p65 complex
in the cytoplasm or due to the ability of I
BR to remove preformed
NF-
B complexes on DNA, as was demonstrated for I
B
and
Bcl-3 in in vitro studies
(44) . Each I
B protein
usually has a unique spectrum of activity for inhibiting DNA binding by
Rel homodimers and heterodimers. The true physiological partner(s) of
I
BR remains to be identified. Differential expression of I
BR
in epithelial cells raises a question whether I
BR is involved in
special functions of these cell types. For example, is I
BR
involved in integrating proliferating/differentiating signals that are
called into action during re-epithelialization of the lung following
injury? Identification of the physiological partner(s) of I
BR will
help elucidate the physiological function of I
BR.
/EMBL Data Bank with accession number(s) U16258.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.