From the Institute of Molecular Medicine, Dr. H.-L.
Tsai Memorial Laboratory, College of Medicine, National Taiwan
University and § Institute of Biological Chemistry, Academia
Sinica and the
Institute of Biochemical Sciences, College of
Science, National Taiwan University, Taipei, Taiwan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcription factor C/EBP has been known to
regulate a wide array of genes including those involved in the
acute-phase response. One of the molecular mechanisms underlying
transcription activation by C/EBP
is through protein-protein
interaction with other transcription factors. Here we report the
identification and characterization of physical and functional
interactions between C/EBP
and heterogeneous nuclear
ribonucleoprotein (hnRNP) K. This interaction results in the repression
of C/EBP
-dependent trans-activation of the agp gene. Footprinting assays indicate that hnRNP K cannot
bind to the promoter region of agp gene or interfere with
the binding of C/EBP
to its cognate DNA site. Furthermore,
agp gene activation by the synergistic interaction of
Nopp140 and C/EBP
is abolished by hnRNP K. The kinetics of
appearance of C/EBP
-hnRNP K complex in the nuclear extract after
initiation of acute-phase reaction indicates that hnRNP K functions as
a negative regulator of C/EBP
-mediated activation of agp
gene.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C/EBP (also called AGP/EBP, NF-IL6, IL6-DBP, CRP2) (1-5)
belongs to the C/EBP transcription factor family which includes C/EBP
(6), C/EBP
(7), C/EBP
(8), and CHOP (9). C/EBP
is a
key transcription factor involved in induction of genes during
acute-phase or immune response (10). Responding to extracellular
stimuli, C/EBP
may form heterodimers with other C/EBP family members
or interact with other transcription factors such as members of the
NF-
B family (11-13), glucocorticoid receptor (14), AP-1 (15), Sp1
(16), and p53 (17). These interactions may result in
cross-communication between transcription factors of different family
members and thus increase the flexibility of gene regulation through
combinatorial mechanisms.
In eukaryotic cells, nascent RNA transcripts are associated with large, multiprotein complexes called heterogeneous nuclear ribonucleoprotein complexes (hnRNP)1 (18). These hnRNP proteins bind pre-mRNAs and appear to facilitate various stages of mRNA biogenesis such as pre-mRNA processing and mRNA transport from the nucleus to cytoplasm (18). Among these hnRNP proteins, hnRNP K is known to be the major poly(rC)-binding protein in HeLa cells (19), and possesses an unusual structure comparing with other hnRNP proteins. Nucleic acid binding activity of hnRNP K is not mediated by an RNA-binding consensus sequence, but by three repeats of motifs termed the KH (K homology) domain (20). These repeated motifs also be found in other proteins, including Ri autoantigen (21), fragile-X protein (22), and MER (23), which are all nucleic acid-binding proteins, suggest that KH motif may be involved in nucleic acid binding. The competition experiments revealed DNA rather than RNA to be the preferred ligand for hnRNP K binding in vitro (24). Thus, it is not surprising that hnRNP K has been repeatedly identified as a sequence-specific DNA-binding protein (25-28). Recently, several reports have shown that hnRNP K can bind to a cis-element within the human c-myc promoter and activates c-myc expression (24, 29). Thus, hnRNP K appears to be involved in transcriptional regulation. Direct protein-protein interaction between hnRNP K and some proto-oncogene provides evidence that hnRNP K acts as a docking platform to facilitate molecular interactions (30-32). Thus, in addition to an architectural component of hnRNP complexes, hnRNP K is also involved in other process such as transcriptional regulation and signal transduction.
To systematically search for proteins that interact with C/EBP, rat
liver nuclear extracts were fractionated with anti-C/EBP
immunoaffinity column chromatography and SDS-PAGE followed by LC/MS/MS
analysis. A number of proteins were identified to be putative
C/EBP
-interacting partners. Among them, a phosphoprotein of 140 kDa,
Nopp140, was identified to be interacting with C/EBP
synergistically
in activating agp gene expression (33). In this report, we
describe the identification and characterization of hnRNP K to be
another C/EBP
-interacting protein. This protein-protein interaction
results in the repression of C/EBP
-dependent
transactivation of agp gene. During the acute-phase
reaction, the kinetics of the decrease of hnRNP K-C/EBP
complex
appears to correlate with the increase of agp gene
expression. These results suggest that hnRNP K is a negative regulator
of C/EBP
-mediated agp gene activation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids--
Full-length hnRNP K cDNA was isolated by
reverse transcriptase-polymerase chain reaction from rat liver RNA and
cloned into pCRII T vector (Invitrogen). The cDNA was analyzed by
restriction enzymes mapping and partial sequencing to confirm that it
is cDNA of hnRNP K. The EcoRI fragment from pCRII/hnRNP
K was subcloned into CMV expression vector (pcDNA3, Invitrogen),
and also subcloned into pGEX vector (Pharmacia) for GST fusion protein
production. For the deletion analysis, EcoRI-NdeI
fragment (amino acids 1-380) or EcoRI-HindIII
fragment (amino acids 1-180) were blunt-ended and ligated to
pcDNA3 and pGEX vectors. Other plasmids, AGP-CAT, C/EBP-CAT, and
CMV-Nopp140, and CMV-C/EBP
constructs were as described (33),
AGP/D-CAT was constructed by oligomerized D site of agp gene
ligated to minimal promoter region of agp gene (34).
Deletion mutants of C/EBP
from pRSET vector (Invitrogen) were
created by NcoI/HindIII digestion for C/EBP
-N
(from amino acids 21 to 146) and by PvuII/HindIII
digestion for C/EBP
-P (from amino acids 21 to 265). CMV-Nopp140/BS
was created by ligation of the BamHI/SacI
fragment of Nopp140 cDNA (containing amino acids 1-169) to a CMV
expression vector.
Recombinant Proteins and Antibodies--
Recombinant hnRNP K,
Nopp140, and C/EBP (both full-length and truncated forms) from pRSET
vector were expressed in Escherichia coli BL21 (DE3, pLysS)
and purified by a nickel column. GST-Nopp140 and GST-hnRNP K from the
pGEX vector (Phamacia) were induced in E. coli DH5
.
Rabbit anti-hnRNP K, anti-Nopp140, and anti-C/EBP
antibodies were
produced by immunizing the rabbit with purified recombinant proteins.
The monoclonal antibodies to C/EBP
were as described (2). The
specificities of these antibodies were characterized by Western blot
analysis using liver nuclear extracts. These antibodies were
monospecific and no cross-reactivities could be detected.
Nuclear Extract Preparation, Immunoprecipitation, and Western
Blot--
Nuclear extracts from rat liver were prepared as detailed
elsewhere (3). For immunoprecipitation analysis, 100 µg of liver nuclear extracts were incubated with 5 µg of anti-Nopp140, anti-hnRNP K, or anti-C/EBP antibody in 1 ml of IP buffer (10 mM
Tris, pH 7.5, 200 mM NaCl, 0.1% Nonidet P-40) and mixed
constantly at 4 °C overnight. The immune complexes were reacted with
protein A-Sepharose at 4 °C for 2 h, washed four times with IP
buffer, and resuspended in SDS loading buffer and subjected to
SDS-PAGE. For Western blot analysis, the separated polypeptides were
blotted into Hybond-C membrane (Amersham) using a semi-dry transfer
unit (CBS, Del Mar, CA) at 1 mA/cm2 constant current for
1 h. The membrane was probed with antibody and detected using the
enhanced chemiluminescence kit (Amersham).
Transient Transfection and Chloramphenicol Acetyltransferase (CAT) Assay-- Baby hamster kidney (BHK) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transfection experiments, the cells were plated on 6-cm diameter Petri dish at about 30% confluence and transfected the next day using the calcium phosphate precipitation method. The amounts of CAT reporter and expression vectors used are detailed in the figure legends. pCMV/SEAP (1 µg, which encodes secreted form of alkaline phosphatase, from Tropix) was included in each transfection as an internal control for transfection efficiency. pCMV plasmid DNA, which contains the CMV promoter only, was used to bring the total DNA to 5 µg. 48 h later, the culture supernatants were collected for alkaline phosphatase detection assays (35). The cells were harvested and CAT assays were performed as described (33). The conversion activities were quantitated with image analyzer (Fuji, BAS 1000) and normalized with alkaline phosphatase activity. All transfection experiments were done with duplicates and repeated two to four times. The relative CAT activities were shown as an average of these independent duplicate experiments. The error bars refer to standard deviation.
Gel Mobility Shift and Footprinting Assays--
The AGP-CAT
plasmid that contained the agp gene promoter region (180
to +60) was digested with HindIII, blunt-ended with Klenow
fragment in the presence of [
-32P]dCTP and dNTPs. The
5'-end-labeled DNA fragment was then cut out by XbaI and
purified from agarose gel with Nucleotrap (Macherey-Nagel). The probe
(20,000 cpm/ng) was added to a 20-µl reaction mixture (25 mM HEPES, pH 7.8, 60 mM KCl, 7.5% glycerol,
0.1 mM EDTA, and 1 µg of poly(dI-dC)). Liver nuclear
extracts or recombinant proteins were added and the binding reaction
was allowed to proceed for 1 h on ice. Then DNase I (1 mg/ml,
freshly diluted in 10 mM MgCl2 and 5 mM CaCl2) was added. DNA fragment was digested
at room temperature for 1 min followed by addition of 80 µl of stop
buffer (75 µg/ml sonicated E. coli DNA, 20 mM
EDTA, 0.5% SDS, and 100 µg/ml proteinase K). The samples were then
incubated for 30 min at 65 °C, extracted twice with
phenol-chloroform, and precipitated with ethanol at
70 °C. The DNA
pellets were analyzed by denaturing gel. The gel retardation assays
were performed as described (2). The oligonucleotide of C/EBP
binding motifs was used as probes and labeled with Klenow fragment in
the presence of [
-32P]dCTP.
Protein-Protein Interaction Assay--
Glutathione-Sepharose 8A
beads (Pharmacia) were mixed with 3 µg of either recombinant
GST-hnRNP K or GST-Nopp140 fusion proteins, or GST only, in 500 µl of
phosphate-buffered saline, 1% Triton X-100 on a rotary shaker for 20 min at room temperature. The beads were washed three times with
phosphate-buffered saline and combined with 100 ng of recombinant
full-length C/EBP, C/EBP
-N, or C/EBP
-P in a final volume of
200 µl of binding buffer (phosphate-buffered saline, 0.1% Triton
X-100) and incubated by shaking on a rotary shaker for 2 h at
4 °C. The beads were washed four times with binding buffer. The
bound proteins were eluted by boiling in SDS-PAGE loading buffer and
subjected to SDS-PAGE for Western blot.
RNA Extraction and Northern Blot Analysis--
Total rat liver
RNA was extracted from rat liver as described previously (2). For
Northern blot analysis, 10 µg of total RNA per lane was resolved by
electrophoresis using 1.5% agarose gel containing 2.2 M
formaldehyde. RNAs were transferred to Hybond N membrane (Amersham),
and hybridized with 32P-labeled C/EBP cDNA probe
(2 × 106 cpm/ml).
Affinity Column Chromatography--
A monoclonal anti-C/EBP
antibody was purified with protein G affinity column. The purified
immunoglobulin G (IgG) was then immobilized to protein A-Sepharose
CL-6B (Pharmacia Biotech Inc.) and cross-linked by the cross-linking
agent dithiobis(succinimidyl propionate) (Pierce). IgG of other
monoclonal antibody was used as a nonspecific immunoaffinity column
control. The column was equilibrated with buffer A (50 mM
HEPES, pH 7.9, 20% glycerol, 0.5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride) containing 0.1 M NaCl. Rat
liver nuclear extract (50 mg) was loaded onto a 1-ml column with
constant recirculation of the flow-through fraction for 30 min at
4 °C. The column was then washed with buffer A containing 0.1 M NaCl. Stepwise elution was performed with 3 ml of buffer A containing 0.2, 0.5, and 1 M NaCl followed by 0.1 M glycine buffer, pH 2.7.
LC/MS/MS Analysis of the Purified Protein--
Rat liver nuclear
extracts were purified by anti-C/EBP antibody column chromatography
and SDS-PAGE. The major polypeptide detected by silver staining of SDS
gel was recovered by extracting the gel slice with buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.1% SDS) at 37 °C overnight. The extracted
protein was precipitated with acetone, washed with 70% ethanol,
dissolved in 20 µl of 10 mM Tris-HCl, 0.5 µl of 1 M CaCl2, 0.2 µg of trypsin, and digested for
18 h at 37 °C. Liquid chromatography-tandem mass spectrometry
(LC/MS/MS, quadrupole spetrometer) is used for sequencing of short
peptides with high sensitivity. This analysis was performed by Dr. John
Yates's Lab at the Department of Molecular Biotechnology, University
of Washington, Seattle, WA.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of hnRNP K in a Complex Containing C/EBP--
In
our previous report (33), a number of C/EBP
-interacting proteins
have been identified from anti-C/EBP
antibody affinity column.
Briefly, one of the specific polypeptides retained by the anti-C/EBP
immunoaffinity column and eluted at 0.5 M NaCl has a
molecular mass of approximately 55 kDa in SDS-PAGE. This polypeptide
was eluted from SDS gel and subjected to trypsin digestion followed by
LC/MS/MS analysis (detailed under "Experimental Procedures"). The
sequence of one of the tryptic peptides matched to the rat hnRNP K
(amino acids 377-396, RGSYGDLGGPIITTQVTIPK), a component of
heterogeneous nuclear ribonucleoprotein complex. The full-length cDNA of hnRNP K was isolated by reverse transcriptase-polymerase chain reaction and the recombinant hnRNP K was expressed in E. coli. The predicted open reading frame of hnRNP K cDNA is a
polypeptide of 464 amino acids which correlates well with the purified
55-kDa protein. Western blot analysis with anti-hnRNP K antibody was performed on eluted fractions of anti-C/EBP
affinity column. The
results demonstrated that both hnRNP K and Nopp140 can be retained by
the C/EBP
antibody affinity column and eluted in the 0.5 M NaCl fractions (Fig.
1A). However, C/EBP
was
eluted at pH 2.7 glycine buffer. To further analyze the biochemical
nature for the retention of hnRNP K by the C/EBP
immunoaffinity
column, we performed immunoprecipitation using rat liver nuclear
extracts. Polyclonal antibody to C/EBP
, Nopp140, or hnRNP K, but not
preimmune serum, can bring down C/EBP
from rat liver nuclear extract
(Fig. 1B). These results indicate the co-existence of
C/EBP
-hnRNP K in a complex in the nuclear extract.
|
hnRNP K Functions as a Repressor of C/EBP-mediated
Transactivation of agp Gene--
To further address the possible
functional interaction between hnRNP K and C/EBP
, we performed
transfection experiments using expression plasmids of hnRNP K and
C/EBP
and reporter AGP-CAT. hnRNP K alone does not have any apparent
effect on AGP-CAT activity; in contrast, Nopp140 or C/EBP
could
activate AGP-CAT expression (Fig.
2A). However, when BHK cells
were co-transfected with hnRNP K and C/EBP
, C/EBP
-mediated
activation of AGP-CAT is repressed by hnRNP K in a
dose-dependent manner (Fig. 2A). To delineate the promoter specificity of this repression, we used promoter from the
C/EBP
gene for the transfection assay. Similarly, hnRNP K
can repress C/EBP
-mediated activation of C/EBP
-CAT reporter (Fig.
2B). To further test the involvement of the C/EBP
-binding motif in the repression of the C/EBP
-mediated transactivation of
target gene by hnRNP K, we conducted transfection experiments using CAT
reporter containing the oligomerized C/EBP
-binding motif
(i.e. AGP/D-CAT, Ref. 34). Again, hnRNP K could repress C/EBP
-mediated activation of the artificial reporter (Fig.
2C). Taken together, these results suggest that hnRNP K
functions as a negative regulator for
C/EBP
-dependent genes activation.
|
Direct Protein-Protein Interaction between hnRNP K and
C/EBP--
Results from the previous section suggest that hnRNP K
and C/EBP
may exist in a complex in the nuclear extract. To further characterize their interaction biochemically, we employed GST-hnRNP K
fusion protein as a bait for probing the recombinant C/EBP
(Fig.
3A). Wild-type (WT) or
truncated forms (C/EBP
-P and C/EBP
-N) of recombinant C/EBP
were incubated with GST-Nopp140, GST-hnRNP K, or GST alone. Both
wild-type and C/EBP
-P could bind GST-hnRNP K specifically (Fig.
3A, lanes 10 and 12), however, C/EBP
-N failed to interact with hnRNP K (Fig. 3A, lane 11). The C/EBP
-N
is a bZIP domain-truncated mutant which retains amino acids 21-146, while C/EBP
-P is a leucine zipper-deleted basic amino acid
region-intact mutant which includes amino acids 21-265. Thus, these
results suggest that like Nopp140, the basic amino acid domain of
C/EBP
is important for its interaction with hnRNP K. To further
investigate the physical and functional interactions of C/EBP
and
hnRNP K, we established two deletion mutants (hnk(380) and hnk(180)) of hnRNP K for protein-protein interaction and transient transfection assay. Both deletion mutants could interact with C/EBP
, albeit with
apparent lower affinity than the wild-type protein (hnk(wt)) (Fig.
3B). However, the results from the co-transfection assays showed that the sequential deletions from the C terminus results in
decreasing repressive activity (Fig. 3C). Taken together,
these results suggest that the intact molecule of hnRNP K is essential for full activity of C/EBP
-binding and repression of
C/EBP
-mediated activation.
|
|
The Kinetic Change of Levels of hnRNP K-C/EBP Complex during
Acute-phase Response--
C/EBP
is one of the key transcription
factors responsible for the induction of genes during acute-phase
response. When the animals were treated with LPS, AGP RNA expression
was induced dramatically after 30 min (Fig.
5A). The results of Western
blot analysis showed that the expression of C/EBP
increased only
slightly while hnRNP K remained unchanged (Fig. 5B). Stat 3 (APRF), a known transcription factor induced by LPS treatment of
animals, was used as a control (Fig. 5B). To assess the
levels of hnRNP K during the acute-phase reaction, we analyzed the
kinetics of appearance of the hnRNP K·C/EBP
complex in the nuclear
extracts from normal and LPS-treated rat liver. Both normal and
LPS-treated nuclear extracts were immunoprecipitated with anti-C/EBP
(BR) or anti-hnRNP K (hn K) antibody (Fig. 5C). The level of
C/EBP
precipitated by anti-C/EBP
antibody was about the same in
the nuclear extract of normal and LPS-treated rat liver. However,
C/EBP
precipitated by anti-hnRNP K antibody decreased at 30 min and
increased thereafter. Taken together, these results suggest that the
decrease in the level of hnRNP K·C/EBP
complex correlated with the
induction of the acute-phase response gene (e.g. agp). hnRNP
K·C/EBP
complex may have a negative effect on C/EBP
-mediated
gene activation.
|
Activation of agp Gene by Synergistic Interaction between Nopp140
and C/EBP May be Inhibited by hnRNP K--
Our previous results
showed that Nopp140 could interact with C/EBP
and activate
agp gene synergistically. To test the effect of hnRNP K on
the synergistic activation of AGP-CAT by Nopp140 and C/EBP
, we
performed transfection experiments using expression vectors of Nopp140
and C/EBP
in the presence of increasing amounts of hnRNP K. The
results showed that hnRNP K acts similarly as the dominant-negative
mutant of Nopp140, Nopp140/BS (i.e. hnRNP K could abolish
the synergistic activation of AGP-CAT by Nopp140 and C/EBP
in a
dose-dependent manner) (Fig.
6A). The overexpression of
increasing amounts of C/EBP
or Nopp140 could also overcome the
repression of hnRNP K (Fig. 6B). To further characterize the biochemical relationship of hnRNP K, Nopp140, and C/EBP
, we
performed the glutathione bead pull-down assay. Recombinant C/EBP
was incubated with GST-Nopp140 in the presence of increasing amounts of
recombinant hnRNP K (Fig. 6C). hnRNP K can abolish the
interaction of Nopp140 and C/EBP
in a dose-dependent
manner. There is good correlation between the disruption of Nopp140 and
C/EBP
complex formation by hnRNP K and the repressive effect of
hnRNP K on the synergistic activation of AGP-CAT by Nopp140 and
C/EBP
.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C/EBP is a key transcription factor responsible for regulating
genes involved in inflammatory and acute-phase responses. Responding to
extracellular stimuli, C/EBP
may cooperate with other transcription
factors in activating its target genes (11-14). Established results
indicate that C/EBP
interacts with a number of transcription factors
physically and functionally (11-17). Several lines of evidence showed
that there are physical and functional interactions between hnRNP K and
C/EBP
. 1) hnRNP K was retarded by anti-C/EBP
antibody affinity
column (Fig. 1). 2) C/EBP
can be immunoprecipitated by anti-hnRNP K
antibody from the nuclear extract (Fig. 1). 3) hnRNP K could repress
C/EBP
-mediated gene activation in a C/EBP
-binding motif dependent
manner (Fig. 2). 4) Direct interaction between hnRNP K and C/EBP
(Fig. 3). 5) The co-existence of hnRNP K and C/EBP
in the complex
formed with C/EBP
-binding motif (Fig. 4).
hnRNP K was first discovered as a component of the hnRNP particle (18).
Recently, hnRNP K has been identified as a DNA-binding factor involved
in transcription regulation. hnRNP K has been identified as human
c-myc CT-element binding protein. Transfection and in
vitro transcription assays indicated that hnRNP K could activate
gene expression in a CT-element dependent manner (24, 28, 29). Thus
hnRNP K functions as a transcription factor when it binds to the
CT-element of c-myc promoter. Using G-rich oligonucleotides
derived from catalase gene silencer element as probe for binding
screening of the expression cDNA library, hnRNP K was isolated from
a rat hepatoma cell line (26, 36). cDNA encoding a 65-kDa
B-motif binding phosphoprotein had been cloned and identified to be
the murine homolog of human hnRNP K (27). It was shown that hnRNP K
could bind to the
B-motif in a sequence-specific manner.
Furthermore, nuclear protein H16, a simian homolog of human hnRNP K,
binds specifically in vitro to the late coding SV-40 virus
DNA strand in the region of transcription control without binding to
the complementary strand (25, 37, 38). Collectively, these observations
provide evidence that the hnRNP K protein is involved in regulation of
gene expression by binding to the specific DNA motif of its target
gene. In this report, we provide evidence that hnRNP K can participate
in gene regulation through protein-protein interaction with C/EBP
without binding to the specific DNA sequence (39).
We have demonstrated two distinct C/EBP-containing complexes in rat
liver nuclear extract. One of these complexes, C/EBP
-Nopp140, functions as an activator, while the other, C/EBP
-hnRNP K, functions as a repressor for C/EBP
-dependent gene transcription.
hnRNP K functions as a dominant-negative regulator by disrupting the synergistic interaction between Nopp140 and C/EBP
through complex formation between Nopp140 and C/EBP
(Fig. 6). During the acute-phase reaction, the decrease of hnRNP K·C/EBP
complex coincides with the
increase of AGP mRNA (Fig. 5). These results together with those of
transfection assays, suggest that the hnRNP K·C/EBP
complex may
serve as a negative homeostatic regulator of agp gene expression.
One of the possible mechanisms of repression by hnRNP K may be the
block of functional transcription preinitiation complex formation. In
our previous report (33), we suggest that Nopp140 may function as a
coactivator by interacting with both C/EBP and TFIIB. We have also
identified a dominant-negative mutant, Nopp140/BS, which failed to
interact with TFIIB but still could interact with C/EBP
(33).
Co-transfection of Nopp140/BS with Nopp140 and C/EBP
abolished the
synergistic activation of the agp gene by Nopp140 and
C/EBP
. The dominant-negative function of Nopp140/BS may be
substituted by hnRNP K for blocking the synergistic activation of the
agp gene by C/EBP
and Nopp140 (Fig. 6). These results
imdicated that interruption of the proper link between C/EBP
and
general transcription factors or other components of the transcription
machinery might be a mechanism of repressed C/EBP
function. Although
there is no direct evidence to suggest that the mechanism for
inhibition of these two factors is the same. Mutational analysis of
hnRNP K indicates that amino acids from 380 to 464 are essential for
full C/EBP
binding. The decreasing of C/EBP
binding activity of
sequential deletions from the C terminus of hnRNP K correlates with the
diminishing of their repressive activity. Thus, intact hnRNP K is
essential for full C/EBP
binding and repressive activity. The
involvement of other repressors in the hnRNP K-mediated inhibitory
effect may also exist. For example, a novel hnRNP K-interacting
protein, Zik1, has been identified as a transcriptional repressor and
the ZiK1-binding region of hnRNP K has been identified between amino
acid 209 to 307 (40).
The molecular mechanisms of kinetic change in the levels of hnRNP
K·C/EBP complex during the acute-phase reaction are unclear. Recently, an interleukin-1-responsive serine/threonine kinase has been
described to associate and phosphorylate hnRNP K (41). Post-translational modification of hnRNP K by a kinase (i.e.
interleukin-1-responsive kinase) may affect the stability and activity
of the hnRNP K·C/EBP
complex. The level of hnRNP K·C/EBP
complex was decreased by 30 min after the initiation of the acute-phase
response and was restored gradually by 60 min. As a control, no
apparent change of the levels of Nopp140·C/EBP
complex was
observed (data not shown). The transcriptional activity of
Nopp140·C/EBP
complex may be modulated by phosphorylation of
Nopp140 during the acute-phase reaction.2 In addition to the
post-translational modifications, alternative splicing forms of
hnRNP K have been reported. These derivatives may also be involved
in transcriptional regulation of the agp gene (42).
Two repressors, nucleolin and hnRNP K, have been identified to be
involved in the regulation of the agp gene. Nucleolin can bind to a specific DNA motif and inhibit agp gene expression
(43). While the repressor function of hnRNP K is mediated by its
interaction with C/EBP. hnRNP K may have important homeostatic
function by maintaining the basal activity of C/EBP
-inducible genes
under normal conditions. We have also demonstrated that hnRNP K can interact with C/EBP
and repress C/EBP
-mediated activation of target genes (data not shown). During the acute-phase reaction, as
contrary to the increase of C/EBP
, C/EBP
is decreased (44). Thus,
it is likely that the complex of C/EBP
and hnRNP K plays somes role
for regulating genes before the initiation of the acute-phase response.
Taken together, these results suggest that hnRNP K may be involved in
the regulation of genes induced by transcription factors of C/EBP
family.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. K. Eng, A. L. McCormack, and J. R. Yates, Department of Molecular Biotechnology,
University of Washington, Seattle, WA, for LC/MS/MS analysis to
identify hnRNP K as the C/EBP-interacting protein.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Council Grants NSC87-2311-B001-101 (to C. J. C) and NSC86-2312-B002-002 (to S. C. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Tel.: 011-886-2-356-2982; Fax: 011-886-2-321-0977.
1 The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein K; PAGE, polyacrylamide gel electrophoresis; LC/MS, liquid chromatography/mass spectroscopy; CMV, cytomegalovirus; GST, glutathione S-transferase; BHK, baby hamster kidney; WT, wild-type; CAT, chloramphenicol acetyltransferase; LPS, lipopolysaccharide.
2 L-H. Miau, C-J. Chang, and S-C. Lee, preliminary results showed that protein kinase A could phosphorylate Nopp140 and enhance its transcriptional activity.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|