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J Biol Chem, Vol. 273, Issue 50, 33561-33565, December 11, 1998
B Activity*
§,,,
From the Department of Cell Biology, Amgen, Inc.,
Thousand Oaks, California 91320 and the ¶ Department of
Microbiology and Immunology, Baylor College of Medicine, Houston, Texas
77030
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Nuclear factor The Rel/NF- Yeast Two-hybrid Screening--
Yeast single-copy plasmids pPC62
and pPC86 (6) were kindly provided by Dr. D. Nathans; the
yeast-selectable marker genes LEU2 and TRP1 were
swapped between these two plasmids, resulting in a GAL4 transcriptional
activation domain vector containing the LEU2 selectable
marker (designated pTA), and a GAL4 DNA-binding domain vector carrying
the TRP1 selectable marker (designated pDB). DNA encoding
amino acids 2 to 300 of c-Rel was cloned into the GAL4 DNA-binding
domain vector pDB, designated pDB-c-Rel, and used as bait to screen a
human B lymphocyte cDNA library (CLONTECH). Positive yeast clones were selected by colony filter Plasmids--
pCI-PPX was constructed by inserting human PPX
cDNA into the pcDNA3 expression vector. A PCR-based procedure
was employed to insert an XbaI site and a hemagglutinin (HA)
tag (YPYDVPDYASL) at the 5'-end and a NotI site at the
3'-end of full-length PPX cDNA. The resulting PCR products were
subcloned into the expression vector pCI-neo (Promega) between the
corresponding restriction sites and designated pCIneo-HA-PPX. PPX
mutants were constructed using the QuickChange site-directed
mutagenesis kit (Stratagene) to substitute leucine, glutamate, or
lysine for the arginine at amino acid 235 in the phosphatase domain
(designated PPX-RL, PPX-RE, and PPX-RK, respectively) of the
pCIneo-HA-PPX construct. The expression plasmids containing c-Rel
(pRSV-c-Rel), NF- Cell Culture, Transfections, and CAT Assays--
293T cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Cells were plated the day before transfection at a
density of 2 × 106 cells per 100-mm dish. 293T cells
were cotransfected with expression plasmids as indicated without or
with the pVA1 plasmid containing the adenovirus VA1 RNA gene to enhance
transient protein expression as described (7), using the calcium
phosphate precipitation protocol (Specialty Media). Cell extracts were
prepared 48 h after transfection, and chloramphenicol
acetyltransferase (CAT) assays were performed as described previously
(4).
In Vivo Association Assay--
293T cells were transfected with
the expression vectors as described above. Whole-cell lysates were
prepared, immunoprecipitated with anti-c-Rel, anti-NF- In Vitro Association Assay--
The GST-c-Rel, GST-p50,
GST-RelA, GST-p53, and GST-c-Jun fusion proteins were expressed in
bacteria and recovered on glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech) according to the manufacturer's instructions.
Whole-cell lysates of HA-PPX transfected 293T cells were passed through
glutathione-Sepharose columns preloaded with each of the GST fusion
proteins. The columns were washed with phosphate-buffered saline, and
the bound proteins were eluted with glutathione elution buffer. HA-PPX
protein was detected by Western blotting using an anti-HA mAb.
Northern Blot Analysis and Fluorescence in Situ Hybridization
(FISH)--
Poly(A)+ RNAs from human and mouse tissues
were obtained from CLONTECH. Each sample (2 µg)
was denatured and electrophoresed on a 1.2% agarose gel containing
formaldehyde and then transferred to a nylon membrane (Amersham
Pharmacia Biotech) in 20 × SSPE. PPX cDNA was labeled with
[32P]dCTP to a specific activity of 108
dpm/µg, and the membranes were hybridized with the PPX cDNA probe at high stringency. FISH was performed as described previously (8).
Electrophoretic Mobility Shift Assay (EMSA)--
EMSA was
carried out with a 32P-labeled DNA probe containing the
NF- The yeast two-hybrid system was used to identify proteins that
interact with c-Rel. The coding sequence of c-Rel cDNA was cloned
into a modified yeast single-copy plasmid GAL4 DNA-binding domain
vector pDB (Fig. 1A). The
resulting plasmid, pDB-c-Rel, was used as a bait to screen a human B
cell line cDNA library. Two positive clones were isolated by a
direct yeast colony filter 
B (NF-B) and the Rel
family of proteins are pleiotropic transcription factors that play
central roles in the immune and inflammatory responses, as well as
apoptosis. Here, we identified a serine/threonine protein
phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that
specifically associated with c-Rel, NF-B p50, and RelA. The amino
acid sequences of human and mouse PPX are 100% identical, and the PPX
gene was mapped to human chromosome 16 p11.2. Overexpression of PPX,
but not catalytically inactive PPX mutants, stimulated the DNA-binding
activity of c-Rel and activated NF-B-mediated transcription. These
results suggest that PPX is a novel activator of c-Rel/NF-B.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
B1 family
of transcription factors includes RelA (also called NF-B p65), RelB,
c-Rel, NF-B1 p50 (also called NF-B p50), and NF-B2 p52 and is
involved in immunological responses, cellular proliferation, and
programmed cell death (1, 2). Rel/NF-B family members share the
300-amino acid Rel homology domain in their amino-terminal regions.
They activate gene expression by binding to B sites via the
DNA-binding domain located within this Rel homology region. Although
the regulation of Rel/NF-B by its inhibitor IB has been
extensively studied, control of the phosphorylation state of
Rel/NF-B remains unclear. Previously, we showed that c-Rel and RelA
are involved in CD28-mediated signal transduction (3, 4). We also
showed that c-Rel is both constitutively phosphorylated in T cells and
that it is also inducibly phosphorylated following T-cell receptor plus
CD28 costimulation in T cells (3). c-Rel knockout mice exhibit profound
defects in T-cell function, including lymphokine (interleukin 2, interleukin 3, and granulocyte macrophage-colony stimulating factor)
secretion and the T-cell proliferative response to T-cell receptor plus
CD28 costimulation (1). To further study the signaling pathway leading
to c-Rel activation, we searched for c-Rel interacting kinases or
phosphatases using the yeast two-hybrid system. Here we identify the
serine/threonine protein phosphatase X (PPX; also called protein
phosphatase 4 (PP4)) that specifically associated with c-Rel and
activated NF-B-mediated transcription.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-galactosidase assay as described (6), and yeast DNA was recovered from positive clones and transformed into Escherichia coli. Plasmids
containing cDNA clones were identified by restriction mapping and
characterized by automated DNA sequencing of both strands. Subsequent
two-hybrid interaction analyses were carried out by cotransformation of
plasmids containing the GAL4 DNA-binding (pDB) and transcriptional
activation (pTA) domains into Saccharomyces cerevisiae.
B p50 (pCMVp50), and RelA (pCMVp65), the 6tkCAT and
BLCAT2 reporter plasmids, as well as the GST-c-Rel, GST-p50, GST-RelA,
GST-c-Jun fusion plasmids have been described previously (3-5, 7). The
HA-tagged human Rab8 plasmid (designated pCIneo-HA-Rab8) was
constructed by the PCR technique.
B p50,
anti-RelA, or anti-c-Fos antibody, and the HA-tagged PPX or Rab8
protein was detected by Western blotting using an anti-HA mAb as
described previously (7). A double-cycle immunoprecipitation was
performed to dissociate the protein-protein complexes as described (3,
5) with some modifications. After transfection, 293T cells were starved
for 1 h at 37 °C in labeling medium (methionine- and
cysteine-free Dulbecco's modified Eagle's medium containing 1 mM -mercaptoethanol and 2% dialyzed fetal bovine
serum), then incubated for 12 h at 37 °C in 2 ml of labeling
medium containing [35S]methionine and
[35S]cysteine (Amersham) (each at 100 µCi/ml). Cell
lysates were prepared and immunoprecipitated with antibodies against
c-Rel, NF-B p50, RelA, or c-Fos proteins. Then, the
immunoprecipitates were washed and resuspended in disruption buffer,
boiled, immunoprecipitated again with an anti-HA mAb, and subjected to
SDS-polyacrylamide gel electrophoresis (10%) and autoradiography.
B binding motif (the double-stranded oligonucleotides
encompassing the NF-B consensus sequence) using a GelShift assay kit
(Stratagene), according to the manufacturer's instructions.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-galactosidase assay. These yeast
single-copy vectors provide a significant advantage for recovery of
specific cDNA clones after transformation into E. coli.
Both positive clones encoded the protein phosphatase X (PPX; also
called protein phosphatase 4 (PP4)); PPX has been previously
identified, but its function remains unknown (9). To examine the
specificity of its interactions in yeast, the coding sequence of PPX
cDNA was cloned into the yeast vectors pTA and pDB, and each of the
resulting plasmids was cotransformed into yeast with either the vector
alone or other pDB or pTA GAL4 fusion plasmids as listed in Fig.
1B. PPX associated not only with c-Rel but also with NF-B
p50; however, no interaction was detected between this clone and either
IB
or Bcl3 (Fig. 1B), suggesting that PPX may interact
specifically with proteins of the NF-B family and affect their
functions.
View larger version (36K):
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Fig. 1.
Structure of modified GAL4 fusion vectors as
well as interaction between PPX and Rel/NF- B proteins.
A, single-copy GAL4 fusion vectors for yeast two-hybrid
screening. The vector pTA (left) encodes GAL4(TA) expressed
from a constitutively active yeast promoter (PADC1) followed by
a multiple cloning site (MCS), the terminator
(TADC1) of the yeast ADC1 gene (18), and the LEU2
yeast selectable marker. In pTA, cDNAs or cDNA libraries are
inserted into multiple cloning site and expressed as translational
fusions to a cassette consisting of the simian virus 40 large tumor
antigen nuclear localization signal sequence. The vector pDB
(right) encodes GAL4(DB) with a multiple cloning site, the
terminator TADC1, and the TRP1 marker. In pDB,
expression of GAL4(DB)-bait fusion protein is controlled by the strong
yeast promoter (PADC1). Both shuttle vectors contain an
ampicillin resistance gene (Ampr), a bacterial
replication origin (Col E1 Ori), a yeast centromere
(CEN6), and a yeast replication origin (ARSH4),
which is a single-copy vector in yeast. B, interaction
between PPX and Rel/NF-B proteins. Yeast Y190 cells were
cotransformed with expression vectors encoding various GAL4 DNA-binding
domain (DB) and GAL4 transcription activation domain
(TA) fusion proteins. Each transformation mixture was plated
on synthetic dextrose plates lacking tryptophan and leucine.
-Galactosidase activity was assayed by using a standard filter
assay.
The human PPX cDNA described here is almost identical in sequence to that described by Brewis and Cohen (10), with the exception of some minor nucleotide sequence discrepancies and one amino acid difference (threonine versus arginine at position 75). The amino acid sequences of human PPX and mouse PPX (GenBankTM accession numbers AF097996 and AF088911) are completely identical. Northern blot analyses revealed that the PPX transcript (~1.6 kilobases) was expressed at various levels in all of the human and mouse tissues examined (Fig. 2). Using the FISH technique (8), PPX was mapped to human chromosome 16 p11.2 (Fig. 3).
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To determine whether the interactions between PPX and c-Rel or other
NF-B proteins can occur in vitro, whole-cell lysates of
HA-PPX transfected 293T cells were passed through glutathione-Sepharose columns preloaded with the following glutathione
S-transferase (GST) fusion proteins: GST-c-Rel, GST-p50,
GST-RelA, GST-p53, and GST-c-Jun. The bound proteins were eluted and
immunoblotted with an anti-HA mAb. 38-kDa PPX proteins were detected in
the GST-c-Rel-, GST-p50-, and GST-RelA-bound fractions, but not in the
GST-p53- and GST-c-Jun bound fractions (negative controls; Fig.
4A). These results indicate
that PPX specifically associates with c-Rel and other NF-B proteins
in vitro.
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To determine whether interactions between PPX and c-Rel and other
NF-B proteins can occur in vivo, 293T cells were
transiently cotransfected with an HA epitope-tagged PPX expression
plasmid and a c-Rel, NF-B p50, or RelA expression plasmid. As
controls, 293T cells were also transfected with a c-Rel, p50, or RelA
cDNA expression plasmid alone. Cell lysates from transfected cells were immunoprecipitated using antibodies against c-Rel, p50, or RelA.
The immunoprecipitated proteins were subjected to Western blotting
using an anti-HA mAb. The results showed that HA-PPX was
coimmunoprecipitated with antibodies against c-Rel, NF-B p50, and
RelA (Fig. 4B, lanes 2, 4, and
6). As negative controls, HA-PPX was not
coimmunoprecipitated with c-Fos (lane 7); furthermore, c-Rel, NF-B p50, and RelA were not coimmunoprecipitated with the
HA-tagged Rab8 protein (lanes 8-10). The relative
expression levels of HA-PPX (data not shown), c-Rel, p50, RelA, c-Fos,
and HA-Rab8 in the transfected cells was confirmed by Western blotting with either anti-HA mAb or antibodies against c-Rel, p50, or RelA (Fig.
4B, lower panel).
Furthermore, 293T cells were transiently cotransfected with an HA-PPX
expression plasmid and a c-Rel, NF-B p50, or RelA expression plasmid
as described above. Their expression was studied by
[35S]methionine and [35S]cysteine labeling,
and their interactions were analyzed by a double-immunoprecipitation
procedure that eliminates most nonspecific binding (3, 5). As shown in
Fig. 4C (lanes 2, 4, and
6), antibodies against c-Rel, p50, or RelA specifically
immunoprecipitated these NF-B proteins together with PPX. The
coimmunoprecipitated HA-tagged PPX protein was verified by the second
immunoprecipitation using anti-HA mAb. Taken together, these results
indicate that PPX is able to associate with c-Rel, p50, and RelA
specifically in vivo. As negative controls, HA-PPX was not
coimmunoprecipitated with c-Fos (Fig. 4C, lane
7); and c-Rel, NF-B p50, and RelA were not coimmunoprecipitated
with the HA-tagged Rab8 protein (lanes 8-10).
To elucidate the underlying mechanism of PPX-c-Rel interaction, we used
EMSA to examine whether overexpression of PPX could alter the
DNA-binding activity of c-Rel. 293T cells were transfected with either
an empty vector or a PPX expression plasmid alone or cotransfected with
a c-Rel expression plasmid plus either an empty vector or a PPX
expression plasmid (10 µg of each plasmid). Various amounts of
nuclear extracts prepared from the transfected cells were subjected to
EMSA using 32P-labeled oligonucleotides containing the
NF-B-binding sequence as a probe (Fig.
5A). As a negative control, a
similar experiment was performed using a PPX mutant (PPX-RL), instead
of the wild-type PPX (Fig. 5B). We found that overexpression
of PPX stimulated the DNA-binding activity of c-Rel about 4-fold (Fig.
5A), while overexpression of the PPX-RL mutant appeared to
slightly decrease the DNA-binding activity of c-Rel (Fig.
5B).
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To determine whether PPX could stimulate NF-B transcriptional
activity in cells, PPX expression plasmid (1 µg) was cotransfected into 293T cells with the reporter construct 6tkCAT, containing two
NF-B enhancer motifs and a TK promoter to drive the CAT gene (5).
BLCAT2 (5), an enhancerless TK-CAT reporter construct, was also used to
show that PPX interacts specifically with the NF-B enhancer. As
controls, 293T cells were transfected with 6tkCAT and BLCAT2 alone. PPX
activated the CAT activity of 6tkCAT in 293T cells about 5-6-fold,
whereas PPX was unable to activate the CAT activity of BLCAT2 in 293T
cells (Fig. 5C). To ensure that the observed NF-B
activation was due to PPX and not other factors, three PPX mutants were
constructed by substituting the arginine (Arg-235) in the phosphatase
domain with leucine, glutamate, or lysine (designated PPX-RL, PPX-RE,
and PPX-RK, respectively), which should abrogate PPX phosphatase
activity. Cotransfection of the PPX mutant constructs with 6tkCAT in
293T cells showed that PPX-RL, PPX-RE, and PPX-RK all failed to
activate NF-B-dependent CAT activity (Fig.
5C). These results suggested that the observed stimulation
of NF-B-dependent transcriptional activity requires functional PPX.
Although protein phosphatase 2A shares a good deal of homology with PPX
(~66% identity); protein phosphatase 2A has been suggested to be
able to inactivate IB kinase and inhibit activation of Rel/NF-B
(11-13). In contrast, PPX appeared to act on Rel/NF-B proteins
directly and activate Rel/NF-B-mediated transcription in
vivo. The fact that PPX stimulated the DNA-binding activity of
c-Rel and activated the NF-B enhancer activity suggests that PPX may
activate NF-B through augmentation of c-Rel activity. While IB
regulation has been well documented, this novel and intriguing
regulation of Rel/NF-B suggests that the control of Rel/NF-B
signaling pathways is far more complex than previously thought. Thus
far, however, the mechanism by which PPX activates Rel/NF-B-mediated
transcription is unclear. It is still plausible that PPX may
dephosphorylate and subsequently activate other c-Rel-associated transcription factors or other kinases regulating IB
(e.g. IB kinases or MEKK1). In fact, an unidentified
phosphatase has been found in the IB kinase complex (14). Taken
together, our results identify a protein phosphatase that may be an
important activator of Rel/NF-B signaling. Further investigation of
the target(s) of this dephosphorylation event is necessary to
understand the mechanism governing the control of Rel/NF-B in cell
proliferation and apoptosis.
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ACKNOWLEDGEMENTS |
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We are grateful to Nancy Rice for
anti-Rel/NF-B antisera; L. Antonio for DNA sequencing; Carol Chang,
Susan Lee, and Hong Gan for technical assistance; Mary Lowe for
secretarial assistance; W. Boyle, R. Bosselman and L. Souza for support.
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FOOTNOTES |
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* This work was supported by Amgen, Inc. (to M. C.-T. H.), National Institutes of Health Grants RO1-AI38649 and RO1-GM49875 (to T.-H. T.), and National Institutes of Health Predoctoral Fellowship in AIDS Research T32-AI07483 (to K. K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF097996 and AF088911.
§ To whom correspondence may be addressed. Tel. 805-447-6721; Fax: 805-447-1982; E-mail: mhu{at}amgen.com.
Scholar of the Leukemia Society of America. To whom
correspondence may be addressed. Tel.: 713-798-4665; Fax: 713-798-3700; E-mail: ttan{at}bcm.tmc.edu.
The abbreviations used are:
NF-B, nuclear factor B; PPX, protein phosphatase X; HA, hemagglutinin; CMV, cytomegalovirus; mAb, monoclonal antibody; EMSA, electrophoretic
mobility shift assay; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; GST, glutathione
S-transferase; FISH, fluorescence in situ hybridization.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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