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J. Biol. Chem., Vol. 277, Issue 28, 24847-24850, July 12, 2002
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From Gene Regulation, Bone, and Inflammation Research, Eli Lilly and Company, Indianapolis, Indiana 46285
Received for publication, March 28, 2002, and in revised form, May 6, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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We provide evidence of a cross-talk between
nuclear receptor and Ser/Thr protein phosphatases and show that vitamin
D receptor (VDR) interacts with the catalytic subunit of protein
phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a
ligand-dependent manner. PP1c specifically interacts with
VDR but not retinoic acid receptor Vitamin D receptor
(VDR),1 a sequence-specific
ligand-dependent transcription factor belonging to the
family of nuclear receptors, mediates biological actions of
1 In this report, we provide evidence of a cross-talk between VDR and
Ser/Thr phosphatases and show that VDR interacts with PP1c in a
ligand-dependent manner in yeast. Although the association of VDR and PP1c/PP2Ac is ligand-independent in vitro and
in vivo in mammalian cells,
1,25(OH)2D3 induces the Ser/Thr phosphatase enzymatic activity of these phosphatases. This induction leads to a
rapid and specific dephosphorylation at the Thr-389 residue of p70 S6
kinase (p70S6k), resulting in inactivation of this kinase.
By co-immunoprecipitation, we also demonstrate the presence of a
ternary complex containing VDR-PP-p70S6k, where PP
represents PP1c or PP2Ac. Using rapamycin, which specifically prevents
Thr-389 phosphorylation, we show that the Thr-389-dephosphorylated p70S6k does not efficiently interact with the VDR-PP
complex, suggesting that p70S6k phosphorylation state
modulates its interaction with the VDR-PP complex. Since
p70S6k is critical for G1-to-S phase
transition, our observations provide a molecular basis of VDR
ligand-induced G1 block in colon/epithelial cancer cells.
Yeast Two-hybrid Screening--
The bait plasmid was generated
by introducing a PCR-amplified VDR-LBD fragment (amino acids
89-427) in-frame with Gal4 DNA binding domain into
EcoRI-BamHI sites of the plasmid pGBKT7
(CLONTECH, Palo Alto, CA). Transformation of yeast
was carried out by using the Yeastmaker transformation kit
(CLONTECH). A colony from pGBKT7-VDR-transformed yeast strain PJ69-2A was grown for 18 h in 50 ml of
SD/Trp GST Pull-down Assays--
PP1c deletion mutants Microcystin Affinity and Phosphatase Activity--
T47D and
Caco-2 cells were passaged in phenol red-free, high glucose Dulbecco's
modified Eagle's medium supplemented with 10% charcoal-stripped fetal
calf serum. For microcystin chromatography, Caco-2 or T47D cells were
treated with 1,25(OH)2D3 (10 Analysis of p70S6k Phosphorylation--
Cells were
treated with 1,25(OH)2D3 (10 To gain a better understanding of the mechanism of action of
1,25(OH)2D3, a yeast two-hybrid system was used
to identify proteins that interacted with VDR in the presence of the
ligand. The sequence of VDR harboring the D-E regions of the receptor
(amino acids 89-427) was used to prepare the bait construct
(pGBKT7-VDR), and cDNAs encoding VDR-interacting proteins were
isolated from a 17.5-day mouse embryo library. Upon characterization,
most of the cDNA clones, whose protein products interacted with VDR
in a ligand-dependent manner, were found to be mouse
RXR
and retinoid X receptor
in yeast. Although VDR-PP1c and VDR-PP2Ac interaction is
ligand-independent in vivo,
1,25-dihydroxy-vitamin D3 induces VDR-associated
phosphatase activity. Further, VDR modulation of PP1c/PP2Ac activity
results in a rapid and specific dephosphorylation and inactivation of
their substrate, p70 S6 kinase (p70S6k). Finally, we
demonstrate that the endogenous VDR, PP1c or PP2Ac, and
p70S6k are present in a ternary complex in
vivo, and the interaction of p70S6k with the VDR-PP
complex is modulated by the phosphorylation state of the kinase. Since
p70S6k is essential for G1-S transition, our
results provide a molecular basis of 1,25-dihydroxyvitamin
D3-induced G1 block in colon cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,25-dihydroxy-vitamin D3
(1,25(OH)2D3). VDR heterodimerizes with
retinoid X receptor (RXR), and at the molecular level VDR-RXR
heterodimers induce gene expression via interaction with vitamin D
response elements present in the promoter regions of responsive genes
(1). This mode of action is known as the "genomic action" of VDR.
However 1,25-(OH)2D3 also induces gene
expression by a mechanism distinct from its classical mode of action.
For example, 1,25-(OH)2D3-induced expression of
monocytic differentiation markers CD14 and CD11b in THP-1 cells
requires phosphatidylinositol 3-kinase (PI 3-kinase) via
ligand-dependent interaction of VDR with the regulatory
(p85) subunit of PI 3-kinase (2). Similarly estrogen receptor interacts
with the p85 regulatory subunit of the PI 3-kinase where estrogen
receptor-PI 3-kinase interaction leads to the activation of protein
kinase B/AKT and endothelial nitric-oxide synthase (3). It thus appears
that cross-talk between nuclear receptors and other signal transduction pathways can lead to either induction of gene expression in a nuclear
receptor-responsive element-independent manner or to
extranuclear/non-genomic induction of enzymatic activities that are
physiologically important, for example, in explaining the
vasoprotective effects of estrogen (3). Further, nuclear receptor
ligands (dexamethasone, triiodothyronine, and retinoic acid)
also induce a rapid dephosphorylation of c-Jun N-terminal kinase
independently of their transcriptional activation (4). Therefore,
nuclear receptors appear to have a functional role both outside and
inside the nucleus. Ser/Thr phosphatases are implicated in the
regulation of a wide variety of cellular functions, namely metabolism,
transcription, translation, development, cell growth, and
differentiation (5). There are two major and structurally related
families of Ser/Thr phosphatases, termed PP1 and PP2A. PP1c and PP2Ac
are the catalytic subunits of PP1 and PP2A, respectively, that
associate with various regulatory and target subunits, thereby
generating distinct holoenzymes with unique localizations,
specificities, and cellular functions (6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
medium at 30 °C with shaking at 270 rpm. After
a growth of 0.8 at A600, cells were pelleted
(1000 × g, 5 min) and resuspended in 5 ml of the
medium to give a final concentration of more than 1 × 109 cells/ml. The entire pGBKT7-VDR-transformed PJ69-2A
culture was combined with 1 ml of Y187 cells pretransformed with a
mouse embryo cDNA library in pGADT7 expression vector
(CLONTECH) in a 2-liter sterile flask.
YPDA/kan (50 ml) was added to the flask and incubated for
24 h at 30 °C with swirling (50 rpm) for mating. The mixture was centrifuged (1000 × g, 10 min), and the cells were
resuspended in YPDA/kan (10 ml). Double transformants were screened on
medium lacking tryptophan, leucine, and histidine containing 25 mM 3-amino-1,2,4-triazole in the presence of
1,25(OH)2D3 (1 µM). Positive
clones were tested for 
-galactosidase activity (7).
1PP1c
(amino acids 105-323), 2PP1c (amino acids 177-323), and 3PP1c
(amino acids 1-200) were prepared by cloning corresponding PCR
amplification products into EcoRI-XbaI sites of
pCDNA3 (Invitrogen). For protein expression,
pGEX3X-VDR-LBD-transformed DH5 cells (25 ml) were grown overnight
and used for inoculating LB medium (250 ml). Induction with 0.1 mM isopropyl-1-thio--D-galactopyranoside was
performed at A600 = 0.5, and cells were allowed
to grow for 3 h at 30 °C with shaking. Bacteria were washed
once in cold PBS, resuspended in 5 ml of extraction buffer (PBS, 5 mM EDTA, 1 mM DTT with protease inhibitors),
and sonicated three times for 10 s with 15-s intervals. Following
centrifugation to remove cell debris, bacterial extract was incubated
(2 h) at 4 °C with a 50% slurry of glutathione-Sepharose beads (250 µl) (Amersham Biosciences). For GST pull-down assays, 15 µl of
glutathione bead-bound GST-VDR-LBD or GST were mixed with 5 µl of
in vitro transcribed and translated [35S]methionine-labeled wild type or mutant PP1c.
Reactions were subsequently incubated in 1 ml of 75 mM KCl,
50 mM NaCl, 20 mM Hepes, pH 7.0, 1 mM DTT, 0.1% Triton-X, 10% glycerol for 2 h at room
temperature in the presence or absence of
1,25(OH)2D3 (107 M).
Beads were washed, resuspended in 2× protein loading dye (Invitrogen),
denatured for 10 min at 70 °C, subjected to SDS-PAGE (12%), and
analyzed by autoradiography.
7
M) for 30 min, washed with cold PBS, and scraped in MC
buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 2 mM MgCl2, 2 mM DTT, 10% glycerol,
1 mM phenylmethylsulfonyl fluoride, and 1× mammalian mixture of protease inhibitors (Sigma)). Cells were centrifuged (1000 rpm) at 4 °C for 5 min, resuspended in MC buffer (3 ml), and
subjected to freeze-thaw. Cell extracts were passed through a 21-gauge
needle, and debris was removed by centrifugation (4000 rpm) for
10 min. Protein concentration was measured using BCA reagent (Pierce).
The control sample was preincubated with 5 nM free
microcystin for 1 h at 4 °C. Cell extract (1 mg of protein) was
mixed with 30 µl of microcystin-Sepharose beads (50% slurry) (Upstate Biotechnology, Lake Placid, NY) pre-equilibrated with MC
buffer and incubated overnight with shaking at 4 °C. Beads were
washed with cold MC buffer containing 150 mM NaCl,
resuspended in 10 µl of 2× protein loading dye, and electrophoresed
on a 10% SDS-polyacrylamide gel together with aliquots from the
unbound fraction (20 µg each). 1,25(OH)2D3
was present in all washes and buffer for the treated samples. For
phosphatase activity, cell extracts (600 µg of protein) were
incubated overnight at 4 °C with agarose-conjugated rabbit anti-VDR
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and beads were
washed four times with washing buffer (25 mM Tris-HCl, pH
7.0, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij35) supplemented with 150 mM NaCl and
1,25(OH)2D3 where needed. Beads were
resuspended in washing buffer, and aliquots (40 µl) were used for
phosphatase activity assay or Western blotting. Ser/Thr phosphatase
activity was measured using the Ser/Thr phosphatase assay kit (New
England Biolabs, Beverly, MA).
8
M) or vehicle for 5, 15, or 30 min, rinsed once with cold
PBS containing phosphatase inhibitor mixture I (Sigma), and scraped off
in the same buffer. Cells were centrifuged at 450 × g
for 5 min and resuspended in 5 volumes of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM
DTT with protease and phosphatase inhibitors) for 5 min. Igepal-640 was
added (0.6% final concentration), and cells were vortexed (10 s) and
centrifuged (10,000 × g, 30 s). The supernatant was kept as the cytoplasmic fraction. Protein extracts (30 µg) were
subjected to 10% SDS-PAGE and Western blotting with various p70S6k antibodies (Cell Signaling, Beverly, MA).
Immunoprecipitation of VDR- or PP1c-bound p70S6k was
performed on 1 mg of cytoplasmic extract as described for the
phosphatase assay.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, -, and -
(Fig.
1A). Among other
VDR-interacting protein cDNAs, a clone, H1, was isolated that contained a 2.2-kb insert corresponding to the cDNA sequence of full-length mouse PP1c. Neither the bait nor the fish vector was
active when expressed alone in yeast cells. However, -galactosidase activity in yeast was observed only when pGBKT7-VDR was expressed with
pGADT7-PP1c in the presence but not in the absence of
1,25(OH)2D3 (Fig. 1, A and
C). Ligand-dependent interaction of VDR-LBD with PP1c was also found to be dose-dependent (Fig.
1B), and the interaction was also observed with full-length
VDR but not with RAR and RXR (Fig. 1, C and
D).
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Fig. 1.
Isolation of VDR-interacting proteins.
A, -galactosidase activity of clones expressing
RXR,,-Gal4-AD or PP1c-Gal4-AD and Gal4-VDR-LBD in yeast in the
presence (black bars) or absence (hatched bars)
of 1,25(OH)2D3 (106
M). B, dose-responsive VDR-PP1c interaction.
Yeast cells were cotransformed with pGBKT7-VDR-LBD along with
pGADT7-PP1c. -Galactosidase activity indicating interaction in the
presence of various concentrations of
1,25(OH)2D3 is shown. C and
D, PP1c specifically interacts with wild type VDR. Yeast
cells transformed with pGBT9-VDR (1 and 2),
pGBT9-RAR (3 and 4), or pGBT9-RXR
(5 and 6) and pGADT7-PP1c (1,
3, and 5) or with empty pGADT7 vector
(2, 4, and 6) were plated on
Leu/Trp/His plates in the
presence or absence of 107 M
1,25(OH)2D3 for VDR, arotinoid acid
(TTNPB) for RAR, and 9-cis-retinoic acid for RXR.
D, -Galactosidase assay of clones shown in C.
Data are representative of three independent experiments.
To further explore VDR-PP1c interaction, a GST-VDR-LBD fusion protein
(containing amino acids 90-427) was purified from
pGEX3X-VDR-LBD-transformed bacterial cells using glutathione-Sepharose
beads. In vitro translated [35S]methionine-labeled PP1c was retained by the
glutathione bead-bound GST-VDR-LBD. In contrast to the yeast two-hybrid
results, in vitro interaction between GST-VDR-LBD and PP1c
was found to be ligand-independent (Fig.
2A). No interaction was
observed between PP1c and glutathione-bound GST protein. To identify
the VDR interaction domain of PP1c, deletion mutants of PP1c were
generated and assayed for interaction in GST pull-down experiments.
Deletion of up to 177 amino acids in the N-terminal portion of PP1c did
not prevent binding to VDR-LBD (Fig. 2A). Interestingly the
N-terminal part contains a number of amino acids essential to the
catalytic function of PP1c, suggesting that the integrity of the
catalytic site is not required for interaction with VDR. Deletion of
123 amino acids from the C terminus of PP1c significantly reduced but
did not abolish its binding to VDR (Fig. 2B), suggesting
that although the C terminus of PP1c provides the major interacting
region, similar to its interaction with other regulatory proteins,
multiple regions are involved in VDR-PP1c interaction (Fig.
2B).
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To verify that endogenous PP1c was physically associated with
endogenous VDR, we purified PP1c-associated proteins using
microcystin-Sepharose (an affinity resin for PP1c and PP2Ac) beads (8).
Cell extracts obtained from control or
1,25(OH)2D3 (107
M)-treated Caco-2 cells were incubated overnight at 4 °C
with microcystin-Sepharose beads. Following extensive washing,
microcystin bead-bound proteins were separated by SDS-PAGE and analyzed
by Western blotting using anti-VDR antibodies. A VDR-PP1c interaction was observed in Caco-2 cells that was not dependent on the presence of
the VDR ligand (Fig. 2C, a). Preincubation of
Caco-2 extract with 0.5 nmol of free microcystin resulted in a loss of
detectable VDR and PP1c, suggesting that VDR-PP1c association was
specific (Fig. 2C, b). Further, microcystin
chromatography of control and 1,25(OH)2D3
(107 M)-treated Caco-2 and T47D cell extracts
followed by Western blotting with anti-VDR antibodies showed VDR-PP1c
interaction in Caco-2 but not T47D cells (Fig. 2D),
suggesting that VDR-PP association is cell
context-dependent. Moreover, RXR was not detected in the
VDR-PP complex retained on microcystin-Sepharose beads (Fig.
2D).
The presence of ligand-independent interaction between VDR and PP1c in
GST and microcystin systems but ligand dependence in yeast prompted us
to examine the possibility that 1,25(OH)2D3 induced the enzymatic activity of VDR-bound Ser/Thr protein
phosphatases. A ligand-mediated increase in phosphatase activity could
explain the increase in VDR gene expression in yeast. Therefore, we
measured VDR-associated Ser/Thr phosphatase activity. Anti-VDR antibody could pull down Ser/Thr protein phosphatase enzymatic activity from
Caco-2 cell extracts (Fig.
3A). Further, 30-min treatment of cells with 1,25(OH)2D3 (108
M) resulted in an enhancement of VDR-associated phosphatase
activity (Fig. 3A). The specificity of VDR-associated
Ser/Thr phosphatase activity was demonstrated by incubation of the
VDR-IP reaction with microcystin (6 nM) that
completely abolished the VDR-associated enzymatic activity (Fig.
3A). Microcystin, a cyanobacterial toxin, is a potent
inhibitor of both PP2Ac and PP1c (9). Aliquots from the VDR-IP used to
measure phosphatase activity were subjected to Western blot analysis to
monitor differences in VDR-associated Ser/Thr phosphatases between
control and 1,25(OH)2D3-treated samples. In
immunoprecipitations, VDR antibody could pull down not only PP1c but
also PP2Ac, and identical levels of PP1c and PP2Ac proteins were
detected in control and treated cell extracts (Fig. 3B). These results demonstrate that the increased Ser/Thr phosphatase activity associated with VDR-IP derived from
1,25(OH)2D3-treated cells did not result from
increased recruitment of Ser/Thr phosphatases to the receptor. In
contrast, as expected, 1,25(OH)2D3 treatment of
Caco-2 cells resulted in increased immunoprecipitation of RXR by
anti-VDR antibodies (Fig. 3B). Taken together these data
suggest that 1,25(OH)2D3 increases the
enzymatic activity of VDR-associated Ser/Thr phosphatases in Caco-2
cells.
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A number of cellular mechanisms dependent on Ser/Thr phosphorylation and relevant to 1,25(OH)2D3 action could be affected by increased VDR-bound phosphatase activity. The effect of 1,25(OH)2D3 on G1-S phase transition is of particular interest since Ser/Thr phosphatases complex with a number of key regulators of cell cycle progression. PP2Ac is known to dephosphorylate and inactivate p70S6k (10-12), a Ser/Thr kinase that induces translation of mRNAs containing 5'-terminal oligopyrimidine sequences, and is essential for G1-S progression (12). PP2Ac is also shown to form a complex with p70S6k, thus indicating the presence of a Ser/Thr phosphatase-kinase signaling module inside the cell (10, 11, 13). Further, phosphorylations at Thr-229, Ser-371, and Thr-389 are essential for activation of p70S6k (12). Since 1,25(OH)2D3 induces G1 arrest in epithelial cells (14), we looked for changes at the phosphorylation state of p70S6k after treatment of Caco-2 cells with the VDR ligand. Caco-2 cytoplasmic extracts probed with phospho-Thr-389-specific p70S6k antibodies showed dephosphorylation of p70S6k after 5, 15, and 30-min treatments of cells with 1,25(OH)2D3 (Fig. 3C). In contrast, treatment with 1,25(OH)2D3 did not change either the protein level of p70S6k or the level of phosphorylated p70S6k at Thr-421/Ser-424 or Ser-411 positions (Fig. 3C). The rapamycin-sensitive FRAP/mTOR kinase is implicated in phosphorylating p70S6k at Thr-389 upon serum stimulation, raising the possibility that 1,25(OH)2D3 could act by decreasing FRAP/mTOR activity. Activity of this kinase correlates to its phosphorylation at Ser-2448 (15). Analysis of Caco-2 cell extracts did not reveal any decrease in FRAP/mTOR phosphorylation level using a phospho-Ser-2448-specific antibody (Fig. 3C), suggesting that decreased Thr-389 phosphorylation on p70S6k was not secondary to reduced FRAP/mTOR activity.
Although PP2Ac-p70S6k interaction is well documented (10-12), PP1c-p70S6k interaction has not been reported. As shown in Fig. 3D, p70S6k could be immunoprecipitated using anti-PP1c antibody, suggesting the presence of a PP1c-p70S6k signaling module. We next tested the possibility that VDR-PP1c or -PP2Ac complexes also contain p70S6k, thereby providing an opportunity for PP2Ac or PP1c to dephosphorylate and thus inactivate p70S6k. Anti-VDR antibody could pull down Thr-389-phosphorylated p70S6k in the absence but not in the presence of the ligand (Fig. 3D). Further, p70S6k did not directly interact with VDR since in vitro translated p70S6k was not retained by GST-VDR-LBD bound to glutathione beads (data not shown). Dissociation of p70S6k from VDR under conditions where Thr-389 was mainly dephosphorylated (via activation of associated PP1c or PP2Ac in the complex) suggested that the interaction could be dependent upon the phosphorylation state of p70S6k. To support this hypothesis, Caco-2 cells were treated with rapamycin (20 nM, 45 min), and VDR immunoprecipitates were analyzed by using phospho-Thr-389-specific antibody. Since rapamycin preferentially inhibits FRAP/mTOR, the upstream kinase of p70S6k (16), as expected rapamycin treatment of Caco-2 cells inhibited phosphorylation of p70S6k at Thr-389 (Fig. 3D). This inhibition resulted in a significant dissociation of p70S6k from VDR, supporting the idea that phospho-Thr-389 preferentially interacted with the VDR-PP complex in the absence of the ligand (Fig. 3D). These results suggest that VDR may act as a platform to compartmentalize VDR-PP1c-p70S6k and VDR-PP2Ac-p70S6k complexes to the same intracellular location or bring them in close proximity so that ligand stimulation could result in induction of Ser/Thr phosphatase activity, which in turn may result in dephosphorylation and inactivation of p70S6k. Therefore, in addition to vitamin D-dependent induction of p21 gene expression (17), VDR ligand-mediated dephosphorylation and inactivation of p70S6k may also play a role in 1,25(OH)2D3-induced G1 arrest of colon cancer cells. Alternatively the VDR-PP1c/PP2Ac-p70S6k pathway may contribute to ligand-mediated inhibition of proliferation of epithelial cancer cells that do not show p21 up-regulation after 1,25(OH)2D3 treatment (18).
Epidemiological studies show that vitamin D confers significant
protection against colorectal cancer (19) and increased VDR expression
is observed in cancerous lesions than normal colon (20, 21). Therefore,
as shown in our model (Fig. 3E), it is tempting to speculate that in
colon cancer cells, at least a part of the cytoplasmic complement of
VDR is compartmentalized as VDR-PP1c/PP2Ac-p70S6k signaling
module. Increased VDR levels coupled with low
1,25(OH)2D3 may inhibit the activity of
PP1c/PP2Ac in this complex, thus rendering the associated
p70S6k active by virtue of its Thr-389 phosphorylation.
This is evident from Fig. 3A since VDR-associated Ser/Thr
phosphatase activity is drastically inhibited in the absence of the
ligand. VDR ligand, upon binding to LBD, appears to impart a
conformational change in PP1c/PP2Ac proteins, thus rendering them
active in terms of their enzymatic activity (Fig. 3A), which
is evident by decreased Thr-389 phosphorylation, inactivation, and
dissociation of VDR-associated p70S6k (Fig. 3, C
and D). The final result of this sequence of events is cell
cycle arrest at the G1-S transition state. Accordingly, VDR
ligands inhibited the proliferation of colon cancer cells in
vitro and reduced tumorigenesis in vivo (22, 23).
Finally, since down-regulation of PP2Ac activity is one of the key
steps in cellular transformation (24, 25), our observation that 1,25(OH)2D3 regulates Ser/Thr phosphatase
activity might contribute to elucidating the role of VDR ligands in
controlling growth and differentiation of normal and cancer cells.
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FOOTNOTES |
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* 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. Tel.: 317-433-4961;
Fax: 317-276-1414; E-mail: nagpal_sunil@lilly.com.
Published, JBC Papers in Press, May 29, 2002, DOI 10.1074/jbc.C200187200
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ABBREVIATIONS |
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The abbreviations used are:
VDR, vitamin D
receptor;
RAR, retinoic acid receptor;
RXR, retinoid X receptor;
PP, Ser/Thr protein phosphatase;
1, 25(OH)2D3,
1,25-dihydroxy-vitamin D3;
p70S6k, p70 S6
kinase;
mTOR, mammalian target of rapamycin;
PI, phosphatidylinositol;
LBD, ligand binding domain;
PBS, phosphate-buffered saline;
DTT, dithiothreitol;
GST, glutathione S-transferase;
IP, immunoprecipitation;
FRAP, FK506-binding protein
12-rapamycin-associated protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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) of 1,25(OH)2D3. Incubation of the
radiolabeled protein with GST alone served as a control. The
Input lane represents 20% of the total volume of the crude
lysate used in each reaction. Beads were washed, and bound proteins
were eluted and analyzed by SDS-PAGE and subjected to fluorography for
35S. B, analysis of a C-terminal deletion of
PP1c in the presence or absence of 1,25(OH)2D3.
The experiment was performed as described in A. C, VDR-phosphatase interaction in Caco-2 cells.
1,25(OH)2D3-treated (+) or untreated (