From the Clinical Research Unit, Diabetes Center, Kyoto National Hospital, Fushimi-ku, Kyoto 612, Japan, the § Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606, Japan, and the ¶ Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
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The 14-3-3 protein family has been implicated in
growth factor signaling. We investigated whether 14-3-3 protein is
involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the
immunoprecipitate with anti-14-3-3 antibody at the basal condition.
100 nM insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study
revealed that glutathione S-transferase-14-3-3
fusion
protein directly associates with recombinant IRS-1. Pretreatment of
recombinant IRS-1 with alkaline phosphatase clearly decreased this
association. Because the recombinant IRS-1 was not phosphorylated on
its tyrosine residues, the results suggest that serine/threonine
phosphorylation of IRS-1 is responsible for the association. When the
cells are treated with insulin, phosphatidylinositol 3
-kinase (PI3K)
is supposed to complex either 14-3-3
-IRS-1 or IRS-1. The
14-3-3
-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared
by a sequential immunoprecipitation, first with anti-14-3-3
and then
with anti-IRS-1 antibodies. The specific activity of the PI3K in the
former was approximately half of that in the latter, suggesting that
14-3-3
protein bound to IRS-1 inhibits insulin-stimulated lipid
kinase activity of PI3K in 3T3L1 adipocytes.
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INTRODUCTION |
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Insulin promotes the rapid autophosphorylation of its receptor
-subunits and tyrosine phosphorylation of several cytoplasmic proteins such as Shc, pp60, Gab-1, and insulin receptor substrate (IRS)1 1 and 2. IRS-1 plays a
central role in insulin signaling. The protein contains a pleckstrin
homology domain, a phosphotyrosine-binding domain that binds to
NPXY motif of the insulin receptor
-subunit and multiple
tyrosine residues that are potential phosphorylation sites. The
tyrosine-phosphorylated IRS-1 associates with several Src homology 2 domain-containing proteins including Grb-2, SHP-2, Nck, Fyn, and the
85-kDa subunit (p85) of phosphatidylinositol 3
-kinase (PI3K).
Associations of these molecules with IRS-1 are believed to further
activate downstream signaling systems, including mitogen-activated
protein kinase and PI3K cascades, that promote mitogenic and metabolic
effects of insulin (for review see Refs. 1 and 2).
14-3-3 protein family, first discovered as acidic proteins in the brain, is highly conserved in animals and plants. At least nine mammalian isoforms have been identified, and six are expressed ubiquitously (3). Originally, 14-3-3 proteins have been shown to be functional as regulators of tryptophan and tyrosine hydroxylases as well as protein kinase C (for review see Ref. 4). However, recently 14-3-3 proteins were found to associate oncogene products, including Raf-1 (5-11), Bcr-Abl, Bcr (12, 13), polyoma middle T antigen (14), and cell cycle control proteins such as Cdc25 phosphatases (15). The interaction of Raf with 14-3-3 leads to Raf activation in several in vivo systems (5, 7, 8). Moreover, the importance of 14-3-3 proteins in signal transduction is suggested by the reports that 14-3-3 protein interacts with PI3K (16) and glycoprotein Ib-IX (17). However, both the mechanism and the physiological role of protein-protein interactions mediated by 14-3-3 proteins are still unclear.
The aim of the present study is to investigate whether the 14-3-3 protein is involved in the insulin signaling pathway. We showed that
14-3-3 protein directly binds to IRS-1 and that insulin increases
this binding via a wortmannin-sensitive pathway in 3T3L1 adipocytes. We
also demonstrated that the 14-3-3
protein negatively regulates
insulin-stimulated activity of PI3K.
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EXPERIMENTAL PROCEDURES |
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Materials--
Anti-IRS-1 (SC559), anti-phosphotyrosine (SC508),
anti-14-3-3 (SC628), and anti-GST (SC459) antibodies were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rat recombinant IRS-1 (number 12-128) and anti-p85PI3K antibodies (numbers
05-217 and 06-195) were from Upstate Biotechnology, Inc. (Lake Placid,
NY). [
-32P]ATP was from New England Nuclear
(Wilmington, DE). Oligonucleotides were made by ABI 392 DNA/RNA
synthesizer (Foster City, CA). Biosynthetic human insulin was kindly
provided by Eli Lilly (Indianapolis, IN). Calf intestine alkaline
phosphatase and Pfu DNA polymerase were purchased from
Toyobo (Osaka, Japan) and Stratagene (La Jolla, CA), respectively.
pGEX-2T vector and glutathione-Sepharose 4B beads were from Pharmacia
Biotech Inc. (Uppsala, Sweden). pCRII vector was from Invitrogen (San
Diego, CA). 14-3-3
cDNA was kindly gifted from Dr. Watanabe
(Hokkaido University School of Medicine, Sapporo, Japan).
Difluorophosphonometyl phenylalanine (difluoroPmp) was kindly gifted
from Dr. Burke, Jr (NCI, NIH, Bethesda, MD). Fetal calf serum and cell
culture reagents were purchased from Life Technologies, Inc. Donkey
anti-rabbit horseradish peroxidase-conjugated antibody and ECL
chemiluminescent kit were from Amersham Corp. Other reagents were from
Sigma and Attest, Inc. (Kyoto, Japan).
Cell Culture and Preparation-- 3T3L1 cells were purchased from ATCC (Rockville, MD) and were grown and differentiated as described previously (18). After 6 h of serum starvation, cells were stimulated with insulin for 3 min at 37 °C and then immediately frozen with liquid nitrogen. For some experiments, cells were preincubated with 100 nM wortmannin for 20 min. Total cell lysates were prepared by a solubilization in 0.5 ml/10-cm dish of ice-cold lysis buffer (50 mM Hepes, pH 7.5, 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 0.1 mg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 2 mM sodium orthovanadate) for 30 min at 4 °C. The insoluble materials were removed by centrifugation (20,000 × g for 20 min at 4 °C). Cytosolic fractions were prepared by a suspending the cells in ice-cold lysis buffer without Nonidet P-40 and homogenizing with a Teflon homogenizer for several strokes. The membrane fractions were removed by ultracentrifugation (100,000 × g for 1 h at 4 °C).
Construction of Fusion Protein--
To prepare GST fusion
protein, full-length rat 14-3-3 cDNA was amplified by polymerase
chain reaction using sense primer added to the BamHI site
and antisense primer added to the EcoRI site and cloned into
pCRII vector. The insertion was restricted with both BamHI
and EcoRI and ligated into pGEX-2T vector in-frame. The
plasmid was subcloned into DH5
cells. Fusion proteins were affinity
purified using glutathione-Sepharose 4B beads according to the
manufacturer's protocol. The protein is designated GST-14-3-3
. GST
protein alone was also prepared for a negative control.
Peptide Synthesis-- YMXM-containing peptide, YZPZSGSYZPZS (nonphosphorylated form), was synthesized by an Fmoc-based strategy. Norleucines (Z) were used instead of methionines (M) as described previously (19). Boc-based techniques were utilized for the preparation of the phosphopeptide possessing substitution of phosphotyrosine (pY) or difluoroPmp for tyrosine in the two YZPZ motifs (20, 21). The peptides were purified by preparative reverse phase HPLC. Analysis of the purified products by mass spectroscopy and analytical HPLC demonstrated homogeneity and sequence accuracy of the synthesized peptides.
In Vivo Association Study-- 2 mg of total cell lysates were incubated with 2 µg of the antibody indicated for 16 h at 4 °C in an end-over-end mixer, followed by the addition of protein A/G-Sepharose for the last hour. The Sepharose-bound immune complexes were then collected by centrifugation (10,000 × g for 1 min) and washed four times in ice-cold washing buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40) and once in phosphate-buffered saline. The immune complexes were denatured in Laemmli's sample buffer by boiling for 5 min and then subjected to immunoblotting.
In Vitro Association Study--
To see whether 14-3-3 protein
associates with IRS-1 in vitro, either 2 mg of total cell
lysate of 3T3L1 adipocytes or 400 ng of rat recombinant IRS-1 protein
was incubated with 50 pmol of either GST alone or GST-14-3-3
fusion
protein in 1.0 ml of lysis buffer for 1 h and then with
glutathione-Sepharose 4B beads for another hour at 4 °C. The
Sepharose-bound complexes were collected by centrifugation (10,000 × g for 1 min) and washed four times in ice-cold washing
buffer and once in phosphate-buffered saline. The complexes were
denatured in Laemmli's sample buffer by boiling for 5 min and then
subjected to immunoblotting with anti-IRS-1 antibody.
Alkaline Phosphatase Treatment-- 400 ng of rat recombinant IRS-1 protein was incubated with 15 units of active or heat-inactivated calf intestine alkaline phosphatase in 20 µl of 50 mM Tris-HCl, pH 8.0, for 30 min at 25 °C and then heat-inactivated for 10 min at 70 °C. The recombinant IRS-1 proteins were then subjected to in vitro association study.
Immunoblotting-- Samples were separated by electrophoresis on 7.5-10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) and then incubated with the antibody indicated for 3 h at room temperature. The membranes were washed in T-TBS (TBS containing 0.1% Tween-20) for 30 min, incubated with donkey anti-rabbit or anti-mouse horse-radish peroxidase-conjugated antibody in T-TBS for 60 min, and washed for 60 min in T-TBS, and then the proteins were visualized using an ECL chemiluminescent kit. The band densities were quantified using an image analyzer Quantity One System (PDI, Sunnyvale, CA).
PI3K Assay--
PI3K activity was measured as described
previously with slight modifications (22, 23). Briefly, the
Sepharose-bound immune complexes with the indicated antibody were
resuspended in 50 µl of 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA. The reaction was then
initiated by the addition of 10 µl of 100 mM
MgCl2, 10 µl of 1 mg/ml of phosphatidylinositol, and 10 µl of 100 µM [-32P]ATP (~20 µCi).
After 10 min of incubation at 25 °C, the reaction products were
analyzed by thin layer chromatography on silica gel plate followed by a
BAS-2000 (Fujifilm, Tokyo, Japan) detection. To measure the PI3K
activity in Fig. 4, 2 mg of total cell lysate was first
immunoprecipitated with 2 µg of anti-14-3-3
antibody, and then
half of the supernatant was immunoprecipitated with 2 µg of
anti-IRS-1 antibody. To eliminate the influence of each antibody on the
activity of PI3K, 2 µg of anti-IRS-1 and anti-14-3-3
antibody were
added to the first and the second immunoprecipitate, respectively,
before the assay.
Statistical Analysis-- Results are expressed as the means ± S.E. All comparisons were made using a two-tailed t test. p < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION |
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To investigate whether 14-3-3 protein is involved in the
insulin signaling pathway, total cell lysates of 3T3L1 adipocytes were
immunoprecipitated with anti-14-3-3
antibody, and the immune complexes were subjected to immunoblots with antibodies against molecules implicated in insulin signaling. We found that a significant amount of IRS-1 was immunodetected in the immunoprecipitate at the
basal condition. 100 nM insulin increased 2.5-fold (basal versus insulin-stimulated condition; 5.30 ± 0.41 versus 13.57 ± 1.16 arbitrary units, n = 6, p < 0.01) the amount of IRS-1 in the immunoprecipitate
(Fig. 1A, lanes 1 and 2). The increase was detected very rapidly after insulin
treatment, reaching to the plateau at 1 min (data not shown). The
effect of insulin was abolished when the cells were pretreated with 100 nM wortmannin (Fig. 1A, lane 3).
However, wortmannin did not affect the amount of immunoprecipitated IRS-1 at the basal condition (data not shown). To confirm these results, the total cell lysates were immunoprecipitated with anti-IRS-1 antibody, and the immune complexes were subjected to immunoblotting with anti-14-3-3
antibody (Fig. 1B). Consistent with the
former results, 14-3-3
proteins were immunodetected in the
immunoprecipitate at the basal condition. Insulin increased the amount
of immunoprecipitated 14-3-3
proteins. The effect of insulin was
reduced by wortmannin pretreatment.
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To ensure that 14-3-3 protein associates with IRS-1, in
vitro association experiments were performed using a GST-14-3-3
fusion protein. GST-14-3-3
protein was incubated with total cell lysates of 3T3L1 adipocytes treated with or without insulin, followed by glutathione-Sepharose 4B beads precipitation. The precipitates were
subjected to immunoblotting using anti-IRS-1 antibody (Fig. 2A). The GST-14-3-3
protein
(but not GST alone) associated with endogenous IRS-1 in the cell
lysates at the basal condition. Insulin increased the association by
2-fold. These results are in agreement with the data obtained from
in vivo studies (Fig. 1).
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To further address whether the direct association of 14-3-3 protein
with IRS-1 can occur, in vitro reconstitution experiments were performed using recombinant IRS-1 and GST-14-3-3
fusion protein. We found that the GST-14-3-3
protein directly associates with recombinant IRS-1 (Fig. 2B). Recently, it has been
determined that 14-3-3 protein binds to phosphoserine (pS)-containing
motifs by the peptide binding and peptide mapping analysis (24, 25). Therefore, to test whether phophorylated residues of recombinant IRS-1
are involved in the association with 14-3-3
protein, effects of
alkaline phosphatase on the association were determined. Pretreatment of the recombinant IRS-1 with alkaline phosphatase clearly decreased this association (Fig. 2C). The results indicate that
14-3-3
protein directly binds to IRS-1 via its phosphorylated
residues, probably phosphorylated serine/threonine residues, because
the recombinant IRS-1 that was used for the experiment was not
phosphorylated on tyrosine residues. These in vitro
reconstitution experiments could explain the association of 14-3-3
protein with IRS-1 at the basal condition. However, mechanisms of the
insulin-induced increase in the association of 14-3-3
protein with
IRS-1 remains unknown. It was shown that RSXpSXP,
RXRXXpS and a cluster of serine residues in the
-helix are the binding motifs for 14-3-3 proteins (24-26).
Interestingly, IRS-1 contains six RXRXXS motifs
(but no RSXSXP motifs) in its amino acid
sequence. Four of them are in the phosphotyrosine-binding domain, and
the others are located in the region containing the YXXM
motifs. Moreover, it was reported that IRS-1 is a substrate for serine
kinase of PI3K activated by insulin (27). Taken together with our
results, it is tempting to speculate that IRS-1 binds to 14-3-3
protein via its phosphorylated serine residues such as
RXRXXpS motif, and insulin treatment results in
the activation of serine kinase of PI3K, which in turn further phosphorylates the serine residues of IRS-1, leading to more
association with 14-3-3
protein. This could be a reason why
wortmannin inhibits insulin-induced increase of the IRS-1-14-3-3
complex (Fig. 1). Of course, other molecules downstream from PI3K, for
example, protein kinase B, which is another serine/threonine kinase,
may be involved in the insulin-induced increase of the IRS-1-14-3-3
complex (28). Further study will be necessary to clarify this point.
An alternative possibility is that insulin-stimulated association of
14-3-3 proteins with IRS-1 is mediated by PI3K. It is well known
that tyrosine-phosphorylated IRS-1 upon insulin stimulation associates
directly with the p85 subunit of PI3K through pYMXM and
pYXXM motifs, resulting in the activation of PI3K (2). It
has been reported that insulin induces serine-phosphorylation of p85 by
PI3K itself (27, 29). As expected, p85 was detected in the
immunoprecipitates with anti-14-3-3
antibody mainly upon insulin
treatment and little in the basal condition. This band was decreased in
the presence of wortmannin (Fig.
3A). If 14-3-3
protein
associates with PI3K directly, insulin apparently increases the amount
of IRS-1 immunoprecipitated with 14-3-3
antibody via PI3K.
Therefore, to explore this possibility, we activated PI3K in
vitro without the insulin receptor-IRS-1 system. pYMXM-
or difluoroPmpMXM-containing peptide was incubated with the
cytosolic fraction of untreated 3T3L1 adipocytes, and the mixture was
immunoprecipitated with anti-p85 antibody (20, 30). The immunoabsorbed
PI3K was further phosphorylated by adding ATP and manganese and then
subjected to in vitro association experiments with
GST-14-3-3
protein. Although both pYMXM- and
difluoroPmpMXM-containing peptide activated the lipid kinase
activity of PI3K (Fig. 3B) and also increased phosphorylation of p85 (Fig. 3C), neither pYMXM-
nor difluoroPmpMXM-containing peptide increased the
association of GST-14-3-3
protein with PI3K in the absence of IRS-1
molecule (Fig. 3D, upper panel). It is thus
unlikely that 14-3-3
protein directly associates with PI3K in 3T3L1
adipocytes, accounting for the insulin-stimulated increase of IRS-1 in
the immunoprecipitates with anti-14-3-3
antibody. Recently, it was
reported that 14-3-3
protein directly binds to PI3K in human T
lymphocytes (16). The difference from our results may be due to the
tissue, 14-3-3 isoform, or species specificity. It is of interest to
note that human but not mouse p85 of PI3K contains
RSXSXP motif in the breakpoint cluster region homologue domain.
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Because less than half the amount of total IRS-1 associates with
14-3-3 protein when the cells are treated with insulin (Fig. 1A), PI3K may complex either 14-3-3
-IRS-1 or IRS-1. To
address the physiological significance of the 14-3-3
-IRS-1-PI3K
complex, the 14-3-3
-IRS-1-PI3K and IRS-1-PI3K complexes were
separately prepared by a sequential immunoprecipitation (Fig.
4). The total cell lysates were first
immunoprecipitated with anti-14-3-3
antibody (Fig. 4, lanes
1 and 2), and then half of the supernatant was immunoprecipitated with anti-IRS-1 antibody (Fig. 4, lanes 3 and 4). The lipid kinase activity upon insulin stimulation
in the former (14-3-3
-IRS-1-PI3K complex; Fig. 4A,
lane 2) was about 25% of that in the latter (IRS-1-PI3K
complex; Fig. 4A, lane 4, and Table
I). A part of each sample in Fig.
4A was also subjected to immunoblots with anti-IRS-1 (Fig.
4B), anti-phosphotyrosine (Fig. 4C),
anti-p85PI3K (Fig. 4D), or anti-14-3-3
(Fig.
4E) antibody. After insulin stimulation, the amounts of
IRS-1, tyrosine-phosphorylated p185, and p85PI3K in the
first immunoprecipitates (Fig. 4, B, C, and
D, lanes 2) were about 50% of those in the
second immunoprecipitates (Fig. 4, B, C, and
D, lanes 4, and Table I). When normalized by the amount of p85, insulin-induced lipid kinase activity was significantly lower in the first immunoprecipitate compared with that in the second
immunoprecipitate. Both the tyrosine phosphorylation state of IRS-1 and
the association of p85 with IRS-1 were similar in the first and the
second immunoprecipitates. Thus 14-3-3
protein appeared to be a
negative regulator for PI3K in insulin signaling system. Here, the
question is raised as to how 14-3-3
protein regulates PI3K. In
preliminary studies using immunoblots with anti-p110
PI3K
antibody, we found that the ratio of p110
catalytic subunit to p85
regulatory subunit of PI3K was decreased in the first immunoprecipitate compared with that in the second
immunoprecipitate.2 Because
the antibody against the p110
subunit was low in the affinity, this
finding must be further confirmed using different antibodies that work
well in the immunoblotting. However, at this point, it should be
emphasized that PI3K activity is reduced in the 14-3-3
-IRS-1-PI3K
complex when normalized by the amount of p85 subunit.
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The physiological role of protein-protein interactions mediated by
14-3-3 proteins is still under investigations. Recently, 14-3-3 protein
has been shown to be a modulator of kinases including Raf-1, protein
kinase C, and PI3K (4, 16). Furthermore, an importance of 14-3-3 protein as a chaperone as well as an adapter molecule has recently been
proposed (31). Indeed, it was reported that 14-3-3 protein mediates the
interaction of Bcr and Raf and the interaction of A20 protein and c-Raf
by functioning as a bridging adapter molecule (13, 32). Therefore, we
tested whether 14-3-3 protein is acting as an adapter molecule in
between IRS-1 and Raf-1. Total cell lysates of 3T3L1 adipocytes treated
with or without insulin were immunoprecipitated with anti-IRS-1
antibody, and the immune complexes were subjected to immunoblotting
with anti-Raf-1 antibody. However, the results did not support the presence of interaction between IRS-1 and Raf-1 via 14-3-3
protein in the 3T3L1 adipocytes, despite the fact that the interaction between
14-3-3
and Raf-1 protein was clearly observed.2 In
conclusion, in this study we showed that the 14-3-3 protein is another
molecule bound to IRS-1 that modulates PI3K in insulin signaling system
in 3T3L1 adipocytes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Masahiko Watanabe for a gift of
14-3-3 cDNA, Dr. Terrence R. Burke, Jr. for a gift of
difluorophosphonometyl phenylalanine, and the Radioisotope Research
Center of Kyoto University (Kyoto, Japan) for helpful support. We also
thank Michiyo Amaoka and Yumi Tanaka for technical assistance and
Kuniko Fukami for secretarial assistance.
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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: Clinical Research
Unit, Diabetes Center, Kyoto National Hospital, 1-1 Fukakusa-Mukaihata, Fushimi-ku, Kyoto 612, Japan. Tel.: 81-75-641-9161, Ext. 528; Fax:
81-75-645-2781; E-mail: akosaki{at}osk2.3web.ne.jp.
1
The abbreviations used are: IRS, insulin
receptor substrate; PI3K, phosphatidylinositol 3-kinase; GST,
glutathione S-transferase; IP, immunoprecipitation;
difluoroPmp, difluorophosphonometyl phenylalanine; Fmoc,
N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; TBS, Tris-buffered saline.
2 A. Kosaki, K. Yamada, and H. Kuzuya, unpublished observation.
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REFERENCES |
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