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INTRODUCTION |
The rat type 1 vasoactive intestinal polypeptide receptor (VIPR
1)1 is expressed in multiple
tissues throughout rat embryonic development and in the adult rat
(1-3). The expression of VIPR 1 gene is regulated by interactions of
multiple transcription factors with its 5'-regulatory sequences (4-6).
While glucocorticoid receptor and Sp1 protein are required for VIPR 1 gene basal transcription activation (4, 5), VIPR repressor protein
(VIPR-RP), a recently characterized transcription factor, represses
VIPR 1 basal transcription (6).
VIPR-RP belongs to a family of proteins that includes
differentiation-specific element-binding protein (DSEB) (7), the large
subunit of murine activator 1 complex (A1p145) (8), mouse replication
factor C140 (mRFC140) (9), PO-GA (10), and PCRH-REB (11). A common
feature of these proteins is that they all contain a region of about 80 amino acids that shares high identity to bacterial ligases (12, 13).
However, VIPR-RP encodes a much smaller protein than DSEB/A1p145, due
to a single base deletion at amino acid 581 in VIPR-RP that results in
a frameshift and a termination codon at amino acid 657 (6). DSEB was
shown to bind to an enhancer element in angiotensinogen promoter that
mediates the irreversible induction of transcriptional activation
during differentiation of 3T3-L1 adipoblasts to adipocytes (7).
A1p145/RFC forms part of a heteropentameric protein complex that is
essential for DNA replication (14). These observations suggest that
this family of proteins may play a dual role as transcription factor as
well as a component of the DNA replication complex.
We showed in the previous study that VIPR-RP binds to VIPR 1 repressor
element specifically and mediates transcriptional repression of the
reporter plasmid containing four copies of its binding sequence (6).
However, the functional domains responsible for VIPR-RP-specific DNA
binding and transcriptional repression have yet to be defined. In this
study, we have mapped the amino acid sequences required for VIPR-RP DNA
binding by analyzing various regions of VIPR-RP either synthesized
in vitro or expressed as GST fusion proteins, using gel
mobility shift assays. Because many sequence-specific DNA-binding
proteins have modular structure (15), we have determined the
transcription repression domains of VIPR-RP by co-transfection of COS-7
cells with chimeras containing different parts of VIPR-RP fused to GAL4
DNA binding domain, together with a reporter gene containing GAL4
binding sites.
Protein phosphorylation is an important mechanism that modulates the
activity of many transcription factors. Several putative phosphorylation sites for protein kinase A (PKA) and casein kinase II
are present in VIPR-RP. In this study, the functional importance of
phosphorylation of VIPR-RP by casein kinase II (CK-II) and PKA was
investigated. We showed that VIPR-RP is phosphorylated by both kinases
in vitro, and we have identified the Ser and Thr residues
responsible for phosphorylation by mutagenesis studies. Finally, we
demonstrated that phosphorylation by PKA inhibits the ability of
VIPR-RP to repress transcription without affecting it subcellular
localization, and that phosphorylation by CK-II enhances VIPR-RP
transcriptional repression and nuclear localization.
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MATERIALS AND METHODS |
Plasmids--
The reporter plasmid (USATKLUC) containing two
GAL4 binding sites inserted in front of thymidine kinase minimal
promoter linked to luciferase (16) was kindly provide by Dr. V. K. Chatterjee (University of Cambridge, Cambridge, United Kingdom). The
construction of expression vector containing GAL4 DNA binding domain
(pGAL1-147) was described previously (17). The various pGAL-VIPR-RP
expression vectors were generated by insertion of the corresponding DNA
fragments from pCMV-VIPR-RP coding for indicated amino acids into the
polylinker of pGAL1-147. Deletion mutants were generated using ExSite
PCR-based site-directed mutagenesis kit (Stratagene), and point
mutations were made using QuikChange site-directed mutagenesis kit
(Stratagene), following manufacturer's instructions. The green
fluorescence protein (GFP) and VIPR-RP fusion construct (GFP-VIPR-RP)
was generated by inserting the KpnI-EcoRV
fragment from pCMV-VIPR-RP into KpnI-SmaI site of
the GFP-C2 expression vector (CLONTECH). To
construct glutathione S-transferase (GST) and VIPR-RP fusion
protein, the coding region of VIPR-RP was amplified by PCR and cloned
in frame at the EcoRI site of pGEX-4T-1 (Amersham Pharmacia
Biotech) vector. Various deletions of GST-VIPR-RP were made using
restriction enzyme digestion or ExSite PCR-based site-directed
mutagenesis kit (Stratagene). The PKA C-subunit expression plasmid was
kindly provided by Dr. Stanley McKnight (University of Washington,
Seattle, WA).
Cell Culture and Transfections--
COS-7 and F9 cells were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin,
and 10% or 15% fetal bovine serum, respectively. Cells were
transfected by calcium precipitation as described previously (5). Each
DNA construct was transfected in triplicate and tested in at least
three independent experiments. Transfection efficiency was monitored by
co-transfecting thymidine kinase promoter linked to chloramphenicol
acetyltransferase. Forty-eight hours after transfection, cell lysate
was prepared and assayed for luciferase and chloramphenicol
acetyltransferase activity as described previously (5).
In Vitro Transcription and Translation--
Various regions of
VIPR-RP were cloned into BlueScript vector (Stratagene) and were
transcribed from T3 promoter and translated in reticulocyte lysate
using TNT coupled reticulocyte lysate system (Promega). A typical
reaction contains 25 µl of rabbit reticulocyte lysate, 2 µl of
reaction buffer, 20 µM amino acid mixture, 1 µg of DNA
template, 40 units of ribonuclease inhibitor, 10 units of T7 RNA
polymerase, and 1 µl of TranscentTM biotin-lysyl-tRNA
(Promega), in a total volume of 50 µl. The reactions were carried out
at 30 °C for 1 h. Two microliters from each reaction were
boiled in loading buffer and separated on 10% SDS-polyacrylamide gels.
Gels were transferred to nylon membranes and blocked by incubation with
Tris-buffered saline (TBS) containing 0.5% Tween 20 (TBST). The
membranes were incubated with streptavidin-horseradish peroxidase
conjugate in TBST for 45 min, washed four times with TBST, and three
times with TBS. The membranes were then incubated with the
chemiluminescent substrate mixture for 1 min and exposed to Kodak x-ray
film for 2 min.
Gel Mobility Shift Assay--
A 42-base pair oligonucleotide
containing the binding site for VIPR-RP was end-labeled with
[
-32P]ATP using T4 polynucleotide kinase.
Binding reactions were performed in 20 µl of binding buffer (20 mM Tris, pH 7.5, 0.1 mM EDTA, 2 mM
MgCl2, 50 mM NaCl, 1 mM
dithiothreitol, 10% glycerol, and 1 µg of nonspecific competitor
poly(dI-dC)) with either 2 µl of in vitro translated
protein or 1 µg of recombinant VIPR-RP expressed in and purified from
Escherichia coli. Binding was 15 min at room temperature.
The anti-VIPRP-RP antibody was added, and the binding was continued on
ice for an additional 30 min. The reactions were electrophoresed on 4%
non-denaturing polyacrylamide gel, dried, and exposed to x-ray
film for 3-6 h.
In Vitro Kinase Assay--
E. coli B21 cells carrying
fusion protein of GST and various regions of VIPR-RP were grown in 2 ml
of 2× YT medium to an A600 of 0.6-0.8. The
expression of the fusion proteins was induced by 0.5 mM
isopropyl-
-D-thiogalactoside for 2 h. Cells were
collected by centrifugation and resuspended in 500 µl of ice-cold
phosphate-buffered saline containing 100 µg/ml lysozyme. Cells were
lysed by three freeze-thaw cycles. The cell lysate was centrifuged, and
20 µl of a 50% slurry of glutathione-Sepharose 4B (Amersham
Pharmacia Biotech) was added to the supernatant and incubated overnight at 4 °C. The Sepharose beads were sedimented. For CK-II assay, the
beads were washed five times with CK-II buffer (20 mM
MES-KOH, pH 6.9, 130 mM KCl, 10 mM
MgCl2, 4.8 mM dithiothreitol), and resuspended in 50 µl of CK-II buffer. The suspension was then incubated with 10 µCi of [-32P]ATP (NEN Life Science Products, Boston,
MA) and 0.1 milliunit of recombinant CK-II (Roche Molecular
Biochemicals) at 37 °C for 1 h. The beads were then washed five
times with the CK-II buffer and resuspended in SDS-loading buffer. For
PKA assay, the beads were washed five times with PKA buffer (50 mM MES-KOH, pH 6.9, 10 mM MgCl2,
0.5 mM EDTA, 1 mM dithiothreitol, and I mg/ml
bovine serum albumin), and resuspended in 70 µl of PKA buffer. The
suspension was then incubated with 10 µCi of
[-32P]ATP (NEN Life Science Products, Boston, MA) and
0.2 milliunit of recombinant PKA (Roche Molecular Biochemicals) at
30 °C for 1 h. The beads were then washed five times with the
PKA buffer and resuspended in SDS-loading buffer. The samples were
boiled for 5 min, resolved on a 12% SDS-polyacrylamide gel, and
subjected to autoradiography.
Fluorescence Microscopy--
COS-7 cells transfected with
GFP-VIPR-RP or GFP-VIPR-RP mutant expression vectors were fixed 24 h after transfection with 2% neutral buffered formaldehyde (2%
formaldehyde, 20 mM NaPO4, pH 7.4) for 15 min
at 37 °C and washed three times with phosphate-buffered saline.
Slides were then examined with fluorescence microscope.
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RESULTS |
Identification of VIPR-RP DNA Binding Domain--
Because VIPR-RP
does not contain typical DNA binding motifs such as zinc fingers or
basic leucine zipper, as present in many transcription factors, we
sought to identify the region of VIPR-RP that is involved in its
binding to DNA. Various regions of VIPR-RP were transcribed and
translated in vitro (Fig.
1A), and the ability of the
in vitro translated proteins to bind to VIPR-RP recognition sequence was tested in gel mobility shift assays. As shown in Fig.
2B, deletion of the C-terminal
46 amino acids did not affect VIPR-RP DNA binding (lane
1-610). However, when 266 amino acids were deleted from the
C terminus, the truncated protein no longer bound to DNA
(lane 1-390). Deletion up to 367 amino acids
from the N terminus did not have effect on VIPR-RP DNA binding
(lanes 178-656 and 367-656). These
results suggest that the region between amino acids 367 and 610 is
sufficient to confer VIPR-RP DNA binding. To further define the VIPR-RP
DNA binding domain, the region between amino acids 367 and 475 was
expressed in and purified from E. coli as a GST fusion
protein. When the purified recombinant protein was used in gel mobility
shift assay, it generated a mobility retarded band (Fig. 2,
lane 2), whereas no mobility shifted band was
observed in the presence of GST (Fig. 2, lane 1).
Furthermore, the mobility shifted band was competed by unlabeled
homologous oligonucleotide (Fig. 2, lane 3), but
not by oligonucleotide containing point mutations of the VIPR-RP
binding site (Fig. 2, lane 4). Addition of
anti-VIPR-RP antibody resulted in a supershifted band (Fig. 2,
lane 5). Similar experiments were performed using
GST fusion protein containing VIPR-RP amino acids 476-610, no DNA binding activity was observed for this fusion protein (Fig. 2, lane 6). These results suggest that the region
between amino acids 367 and 475 is required for VIPR-RP DNA
binding.
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Fig. 1.
Mapping the DNA binding domain of
VIPR-RP. A, various parts of VIPR-RP were transcribed
and translated in vitro and labeled with
TranscentTM Biotin-lysyl-tRNA. The protein products were
analyzed on SDS-polyacrylamide gels in the lanes indicated, blotted,
and visualized by chemiluminescent detection. B, gel
mobility shift assay using 32P-labeled VIPR-RP binding site
and the in vitro translated protein shown in A.
The DNA-protein complexes are indicated by arrowheads.
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Fig. 2.
Gel mobility shift assay using recombinant
VIPR-RP. The DNA sequence encoding amino acids 367-475 and
476-527 were cloned into pGEX-4T prokaryotic expression vector. The
GST-VIPR-RP fusion proteins was induced by
isopropyl--D-thiogalactoside and purified using
Sepharose 4B. The purified proteins was incubated with
32P-labeled VIPR-RP binding site oligonucleotide and
analyzed on 4% polyacrylamide gel. Lane 1, GST;
lane 2, GST-VIPR-RP(367-475); lane
3, competition with unlabeled wild type oligonucleotide;
lane 4, competition with mutant oligonucleotide;
lane 5, with anti-VIPR-RP antibody;
lane 6, GST-VIPR-RP(476-527). The DNA-protein
complex is indicated by an arrow, and the supershifted band
is indicated by an arrowhead.
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Identification and Characterization of the Transcriptional
Repression Domains--
To localize the transcriptional repression
domain of VIPR-RP, fusion constructs were made between various regions
of VIPR-RP and the GAL4 DNA binding domain (GAL4-DBD, amino acids
1-147). The GAL4-DBD contains signals for dimerization (18) and
nuclear translocation (19) in addition to its specific DNA binding
activity, and shows no transactivation function. Therefore, VIPR-RP
deletions can be analyzed for transcriptional regulation even when
dimerization and nuclear translocation domains are deleted. The
expression plasmids coding for GAL4-DBD fused to various parts of
VIPR-RP were transfected into COS-7 cells together with a reporter
plasmid containing two GAL4 binding sites in front of TK
promoter-luciferase fusion (16). As shown in Fig.
3, deletion of the 279 amino acids from
the C terminus (Fig. 3, construct 1-377)
resulted in loss of transcriptional repression. However, fusion
construct with the deletion of an additional 200 amino acids from the C
terminus (Fig. 3, construct 1-177) was able to
repress the expression of the reporter gene. Similarly, deletion of the
N-terminal 177 amino acids resulted in complete loss of transcriptional
repression (Fig. 3, construct 178-656), but
construct with further deletion (Fig. 3, construct
378-656) showed strong repression function. These results
showed that inclusion of amino acids 178-377 in either N-terminal
(construct 178-656) or C-terminal (construct 1-377) deletion mutant
resulted in loss of transcriptional repression. However, deletion of
this region, as in constructs 1-177 and 378-656, restored repression
function. These results suggest that VIPR-RP contains two independent
transcriptional repression domains located between amino acids 1 and
177 and between 378 and 656, and that the region between amino acids
178 and 377 suppresses VIPR-RP transcriptional repression function. The
latter observation was confirmed by transfecting COS cells with fusion
construct with the deletion of amino acids 178-378. Deletion of this
region resulted in enhanced transcriptional repression activity of
VIPR-RP (Fig. 3, construct 
178-378).
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Fig. 3.
Identification of VIPR-RP transcription
repression domains. Various parts of VIPR-RP were fused to the
yeast GAL4 DBD (GAL1-147). The fusion plasmids were transiently
transfected into COS-7 cells and were assayed for the ability to
repress a target gene. The reporter contains two GAL4 binding sites
upstream of the thymidine kinase promoter-luciferase fusion. Values are
represented as -fold repression over GAL1-147, which was set
arbitrarily as 1.00. Values represent mean from three independent
experiments. The transcription repression domains are shown in
patterned boxes (amino acids 50-101 and
469-527), and the domain that inhibits transcription repression is
shown in gray (amino acids 178-377).
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To further define the amino acid sequences required for VIPR-RP
transcriptional repression, detailed deletions were made within the
regions between amino acids 1 and 177 and between 378 and 656. As shown
in Fig. 3, deletion between amino acids 101 and 177 resulted in a
slight increase in transcriptional repression (Fig. 3,
construct 1-101), and further deletion of the
N-terminal 49 amino acids had little effect on repression activity
(Fig. 3, construct 50-101). Deletion of the
amino acids between 378 and 527 resulted in complete loss of
transcriptional repression (Fig. 3, construct
527-656). However, deletion of the C-terminal 129 amino
acids did not affect repression activity (Fig. 3, construct 378-527). Within the region between amino acids 378 and
527, the C-terminal half (Fig. 3, construct
469-527) conferred transcriptional repression at the same
level as construct 378-527, whereas the N-terminal half (Fig. 3,
construct 378-469) showed much reduced repression activity. These results suggest that the transcriptional repression domains of VIPR-RP is located between amino acids 50 and 101 and between 469 and 527.
VIPR-RP Is Phosphorylated by CK-II in Vitro--
The N-terminal
transcriptional repression domain of VIPR-RP contains two clusters of
acidic amino acids interspersed with Ser and Thr residues (amino acids
D68SDSESEE75, and
S108ETDEDDD115), which correspond to consensus
phosphorylation sites for CK-II (20, 21). We sought to determine
whether phosphorylation by CK-II has any effect on VIPR-RP function.
Initially, we tested whether CK-II can phosphorylate VIPR-RP in
vitro. The GST fusion protein containing either VIPR-RP amino
acids 1-177 or 178-656 were expressed in E. coli, purified
using glutathione-Sepharose, and incubated with recombinant CK-II in
the presence of [-32P]ATP. As shown in Fig.
4A, the fusion protein
containing VIPR-RP amino acids 1-177 was phosphorylated by CK-II,
whereas the fusion protein containing amino acids 178-656 was not
phosphorylated. To test which of the Ser and Thr residues are
phosphorylated by CK-II, site-directed mutagenesis was used to change
Ser-69, -71, and -108, as well as Thr-110, to alanines. As shown in
Fig. 4B, substitution of Thr-110 resulted in about 40%
reduction in VIPR-RP phosphorylation, whereas substitution of Ser-108
alone did not have any effect. When both Ser-69 and -71 were changed to
alanine, VIPR-RP phosphorylation level was dramatically reduced. The
mutant containing all three substitutions at Ser-69/71 and Thr-110 was no longer phosphorylated by CK-II. These results indicate that VIPR-RP
is phosphorylated by CK-II on Ser-69/71 and Thr-110.
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Fig. 4.
VIPR-RP is phosphorylated by CK-II in
vitro. GST fusion proteins contain indicated regions
and point mutations of VIPR-RP, and were expressed in and purified from
E. coli. They were phosphorylated in vitro with
recombinant CK-II, separated on 10% SDS-polyacrylamide gels, and
subjected to autoradiography. Phosphorylated protein is indicated by an
arrowhead. The consensus phosphorylation sites are shown at
the bottom of the figure, and the phosphorylated Ser and Thr
residues are indicated in italics.
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Phosphorylation of the CK-II Sites Enhances VIPR-RP Transcriptional
Repression Function--
To test the effect of phosphorylation of the
CK-II consensus sites on VIPR-RP functions, the same alanine
substitutions for Ser-69/71 and Thr-110 were made in the VIPR-RP
eukaryotic expression plasmid (6). The ability of the mutant VIPR-RP to
repress transcription was then tested by co-transfecting COS-7 cell
with a reporter gene containing four copies of VIPR-RP binding site in
front of TK-luciferase (4FTKLUC). As shown in Fig.
5, co-transfection with wild type VIPR-RP
expression vector resulted in more than 8-fold repression in luciferase
activity of the reporter plasmid, whereas co-transfection with the
mutant VIPR-RP could only repress reporter gene expression a little
over 2-fold (Fig. 5). These results indicate that VIPR-RP containing
defective CK-II phosphorylation sites is a weaker transcriptional
repressor compared with the wild type.
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Fig. 5.
Phosphorylation by CK-II enhances VIPR-RP
transcriptional repression. COS-7 cells were transiently
co-transfected with either wild type or mutant VIPR-RP and a reporter
plasmid containing four copies of VIPR-RP binding sites upstream of
TK-luciferase (4FTKLUC). Forty-eight hours after transfection, cell
extracts were assayed for luciferase activity, represented as -fold
repression over the reporter alone. Values represent mean ± S.E.
from three independent experiments.
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Protein Kinase A Phosphorylates VIPR-RP in Vitro--
Cyclic AMP
(cAMP) mediates hormonal stimulation of a variety of eukaryotic genes
(22). Since most of known cellular effects of cAMP occur via the
catalytic subunit (C-subunit) of cAMP-dependent kinase
(PKA), it is likely that this enzyme mediates the phosphorylation of
factors that are critical for transcriptional response. The presence of
multiple potential phosphorylation motifs (23) in VIPR-RP prompted us
to ask whether this transcription factor is phosphorylated by PKA.
The GST fusion protein containing various parts of VIPR-RP were
expressed in E. coli, purified using glutathione-Sepharose, and incubated with recombinant C-subunit of PKA in the presence of
[-32P]ATP. As shown in Fig.
6A, fusion protein containing
amino acids 1-377 and 178-656 were phosphorylated by PKA, whereas
fusion proteins containing amino acids 1-177 and 378-656 were not
phosphorylated. These results suggest that the phosphorylation sites
for PKA are located between amino acids 178-377. Within this region,
there are five potential Ser and Thr residues that can be
phosphorylated by PKA. To determine which of these residues are
involved in PKA phosphorylation, site-directed mutagenesis was
performed to change each one of these residues to alanine. As shown in
Fig. 6B, Ala substitution of Ser-245 and -361 resulted in
about 50% reduction in VIPR-RP phosphorylation, whereas mutations on
Ser-189, -338, and Thr-372 had no effect. To confirm that Ser-245/361
are responsible for VIPR-RP phosphorylation by PKA, both of these
residues were changed to Ala. As shown in Fig. 6C,
substitution of Ser at both positions 245 and 361 resulted in complete
loss of phosphorylation. These results indicate that VIPR-RP is
phosphorylated by PKA on Ser-245/361.
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Fig. 6.
VIPR-RP is phosphorylated by PKA in
vitro. GST fusion proteins contain indicated regions
and point mutations of VIPR-RP, and were expressed in and purified from
E. coli. They were phosphorylated in vitro with
recombinant C-subunit of PKA, separated on 10% SDS-polyacrylamide
gels, and subjected to autoradiography. Phosphorylated protein is
indicated by an arrowhead.
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Phosphorylation by PKA Inhibits VIPR-RP Transcriptional Repression
Function--
To test whether PKA phosphorylation of VIPR-RP affects
its transcriptional repression activity, we performed transient
transfections in the kinase A-defective F9 teratocarcinoma cells. As
shown in Fig. 7, when co-transfected with
the wild type VIPR-RP alone, the reporter gene 4FTKLUC activity was
reduced 8-fold. Co-transfection with a metallothionein vector
expressing the C-subunit (24) of PKA did not have significant effect on
4FTKLUC activity. Co-transfection of the C-subunit together with
VIPR-RP caused a 2-fold reduction on 4FTKLUC activity, suggesting that
phosphorylation by PKA inhibits VIPR-RP function as a transcriptional
repressor. This observation was confirmed by co-transfecting F9 cells
with VIPR-RP mutant that was defective in the kinase A phosphorylation
motif (S245A/S361A). This mutant alone was able to repress 4FTKLUC
activity to the same level as the wild type VIPR-RP (Fig. 7). However,
co-transfection with the C-subunit expression vector no longer
inhibited the transcriptional repression activity of the mutant VIPR-RP
(Fig. 7).
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Fig. 7.
Phosphorylation by PKA inhibits VIPR-RP
transcriptional repression. F9 cells were transiently
co-transfected with indicated expression plasmids and a reporter
plasmid containing four copies of VIPR-RP binding sites upstream of
TK-luciferase (4FTKLUC). Forty-eight hours after transfection, cell
extracts were assayed for luciferase activity, represented as -fold
repression over the reporter alone. Values represent mean ± S.E.
from three independent experiments.
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Effects of Phosphorylation of CK-II and PKA Sites on VIPR-RP
Subcellular Localization--
To determine the mechanisms by which
phosphorylation of VIPR-RP by CK-II and PKA affects its repression
function, we asked whether phosphorylation by these kinases might have
an effect on the subcellular distribution of this transcription factor. Toward this end, we transfected COS-7 cells with expression vector containing GFP fused to wild type VIPR-RP or mutants containing defective phosphorylation sites for either CK-II or PKA. The expression of the fusion proteins was examined under the fluorescence microscope. As shown in Fig. 8 (A and
B), both the wild type and mutant containing defective
phosphorylation sites for PKA were almost exclusively localized to the
nucleus (Fig. 6, A and B). However, mutant with defective CK-II phosphorylation sites was either localized to both the
nucleus and the cytoplasm, or exclusively in the cytoplasm (Fig.
8C). These results suggest that phosphorylation by PKA does not affect VIPR-RP subcellular localization, whereas phosphorylation of
the CK-II sites enhances VIPR-RP nuclear translocation.
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Fig. 8.
Effects of phosphorylation on VIPR-RP
subcellular localization. COS-7 cells were transfected with
GFP-VIPR-RP wild type (A), GFP-VIPR-RP PKA mutant
(B), or GFP-VIPR-RP CK-II mutant (C). Twenty-four
hours after transfection, cells were fixed, and localization of VIPR-RP
and the mutant proteins were detected by the green fluorescence of GFP.
Both wild type and PKA mutant of VIPR-RP are expressed predominantly in
the nucleus, whereas the CK-II mutant is localized in both the nucleus
and the cytoplasm.
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DISCUSSION |
The experiments described in this study represent the initial
characterization of VIPR-RP functional domains. Our results showed that
amino acids 367-475 are required for specific DNA binding of VIPR-RP
to its binding site in the VIPR 5'-flanking region. Although this
region does not contain any recognizable consensus for characterized
DNA-binding proteins, it contains two predicted 
-helical regions of
14 and 13 amino acids, located between amino acids 413 and 426 and
between 450 and 462. Identical -helical regions are also present in
DSEB DNA binding domain (7). The importance of these putative helixes
in DSEB DNA binding was demonstrated by mutagenesis studies (7). An
alanine to proline substitution within either helix resulted in
complete loss of DNA binding (7). Interestingly, the amino acid
sequences required for DNA binding of both VIPR-RP and DSEB also
contain the region (amino acids 402-474) where this family of proteins share high identity with bacterial DNA ligases. The DNA binding domains
of the DNA ligases have yet to be characterized. However, based on the
high degree of homology, it is possible that this region within the
ligases may be involved in DNA binding.
Our previous study showed that VIPR-RP is able to repress transcription
of a reporter plasmid containing four copies of its binding sequence
when co-transfected into COS-7 cells (6). The evidence in this study
indicates that VIPR-RP contains two separate transcriptional repression
domains, located at the N terminus between amino acids 50 and 101 and
C-terminal to the DNA binding domain between positions 469 and 527. Each domain is capable of transcriptional repression by itself when
fused to the GAL4 DNA binding domain, if amino acids between 178 and 377 are deleted. However, both domains are required for repression function in the presence of this sequence. Many transcription factors
contain multiple activation/repression domains. For example, the
glucocorticoid receptor has two activation domains located in the
glucocorticoid receptor N terminus and C-terminal of the DNA binding
domain (25). Four separate activation domains have been identified in
the thyroid receptor. One domain was localized in the N terminus, and
the other three were identified in the C terminus adjacent to the
ligand binding domain (26).
The activity of many transcription factors is regulated by protein
phosphorylation (27). Some transcription factors bind to DNA in
unphosphorylated form; however, phosphorylation is required for their
transactivation functions. An example of this mechanism is illustrated
by cAMP response element-binding protein. Increases in intracellular
cAMP levels induce phosphorylation of cAMP response element-binding
protein at a serine residue at position 133 by protein kinase A (28),
enhancing its ability to activate transcription without affecting its
intracellular location or DNA binding (28). A second mechanism involves
phosphorylation-induced nuclear translocation of transcription factors
that are present in a latent state in the cytoplasm. For example, a
single phosphorylation site on STAT1 (signal transducers and activators
of transcription), Tyr-701, appears to be both necessary and sufficient
to mediate nuclear translocation, DNA binding activity, and
transcriptional activation by interferon- (29, 30). The presence of
consensus phosphorylation sites for PKA and CK-II in VIPR-RP prompted
us to ask the question whether VIPR-RP is a substrate for these kinases
and what effects phosphorylation by these kinases have on VIPR-RP
function. Our results showed that VIPR-RP was phosphorylated in
vitro by PKA on Ser-245/361 and by CK-II on Ser-69/71 and
Thr-110.
PKA appears to be the major mediator of cAMP responses in mammalian
cells (22). Increased intracellular cAMP levels activates PKA, and
induces transport of the C-subunit into the nucleus (31), where it
mediates phosphorylation of the transcription factors that activate
cAMP-responsive genes. Furthermore, microinjection of the C-subunit
into cells can directly activate transcription of cAMP-responsive gene
c-fos and the gene for vasoactive intestinal polypeptide
(32). Our results showed that phosphorylation by PKA inhibits VIPR-RP
transcriptional repression without affecting it subcellular
localization. Since no cAMP-responsive element is present in the VIPR 1 gene, phosphorylation of VIPR-RP by PKA may provide an indirect
mechanism for cAMP-regulated gene expression. Instead of directly
acting on the cAMP-responsive element, increased intracellular cAMP
levels may activate VIPR 1 gene transcription by inhibition of the
activity of its transcriptional repressor, VIPR-RP.
CK-II is a ubiquitous kinase, which has been shown to be localized in
both the nucleus and the cytoplasm (33) and can be activated following
growth factor stimulation (34, 35). A number of CK-II substrates have
been identified. These include proteins involved in regulation of
transcription, translation, as well as components of signaling pathways
(reviewed in 21). For example, proto-oncogene c-myc was
shown to be phosphorylated within the acidic activation domain by
CK-II, and it was postulated that CK-II mediated phosphorylation of Myc
plays a role in signal transduction of mitogenic stimuli to the nucleus
(36). Although we have not directly tested whether VIPR-RP is
phosphorylated by CK-II in vivo, we demonstrated that in
transfected cells VIPR-RP containing defective CK-II sites was not able
to repress transcription of the reporter gene to the same level as the
wild type VIPR-RP, suggesting that the activity of VIPR-RP can be
modified by phosphorylation of the CK-II sites, either by CK-II itself
or by other kinases that recognized the same phosphorylation site.
CK-II phosphorylation sites were found in many nuclear proteins in the
vicinity of their nuclear localization signals (NLS), including c-Myc
(37, 38), p53 (39, 40), and SV40 T-antigen (41, 42). The average
distance between CK-II site and NLS in these proteins is 23 ± 12 amino acids. It has been shown that phosphorylation by CK-II of a
serine residue near the NLS of SV40 T-antigen enhanced the rate of
nuclear transport of the protein (42). It was postulated that a
conformational change of the NLS, induced by CK-II phosphorylation at
the nearby site, might modulate the affinity of NLS for its presumptive
receptor. The CK-II phosphorylation sites identified in VIPR-RP differ
from these nuclear proteins in that they are not localized near the putative NLS. Three putative bipartite NLS are present between amino
acids 480 and 497, 510 and 527, and 523 and 540, whereas the CK-II
sites are located in the N terminus. Our results indicated that
phosphorylation of the CK-II sites enhances VIPR-RP nuclear localization, because mutant defective in CK-II phosphorylation sites
is partially localized in the cytoplasm. It appears likely that
phosphorylation of the CK-II sites potentiates the ability of VIPR-RP
to repress transcription by ensuring its nuclear localization.
In summary, we have identified functional domains that are important
for VIPR-RP DNA binding and transcriptional repression. We have
demonstrated that phosphorylation by PKA and on CK-II consensus sites
modulates VIPR-RP function through different mechanisms.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Div. of Endocrinology
and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Blvd., D3066,
Los Angeles, CA 90048. Tel.: 310-423-7682; Fax: 815-352-6253; E-mail:
pei@cshs.org.
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