Division of Endocrinology and Metabolism, Departments of 1 Medicine and 2 Anatomy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192 Japan
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ABSTRACT |
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Pancreatic
duodenal homeobox-1 (Pdx1) is a transcription factor, and its
phosphorylation is thought to be essential for activation of insulin
gene expression. This phosphorylation is related to a concomitant shift
in molecular mass from 31 to 46 kDa. However, we found that Pdx1 was
modified by SUMO-1 (small ubiquitin-related modifier 1) in -TC-6 and
COS-7 cells, which were transfected with Pdx1 cDNA. This modification
contributed to the increase in molecular mass of Pdx1 from 31 to
46 kDa. Additionally, sumoylated Pdx1 localized in the nucleus. The
reduction of SUMO-1 protein by use of RNA interference (SUMO-iRNAs)
resulted in a significant decrease in Pdx1 protein in the nucleus. A
34-kDa form of Pdx1 was detected by the cells exposed to SUMO-iRNAs in
the presence of lactacystin, a proteasome inhibitor. Furthermore, the
reduced nuclear sumoylated Pdx1 content was associated with significant lower transcriptional activity of the insulin gene. These findings indicate that SUMO-1 modification is associated with both the localization and stability of Pdx1 as well as its effect on insulin gene activation.
small ubiquitin-related modifier 1; pancreatic duodenal homeobox-1; ribonucleic acid interference
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INTRODUCTION |
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PANCREATIC DUODENAL
HOMEOBOX-1 (Pdx1) is a transcription factor encoded
by a homeobox gene (27, 33, 34). In humans and other
animal species, the embryonic development of the pancreas requires
Pdx1. This role for Pdx1 is supported by the finding of a mutant Pdx1
in an individual with pancreatic agenesis (16, 45).
Furthermore, Pdx1 appears to be essential for transcriptional regulation of a number of specific genes, including insulin (34, 35, 44), somatostatin (27), islet amyloid
polypeptide (2), type 2 glucose transporter
(46), and glucokinase (49), in adult
pancreatic cells. Insulin expression is activated by the binding of
Pdx1 to the A1 and A3/4 elements of rat insulin-1 promoter (26). Conversely, a decline in insulin expression
is observed in cultured -cells exposed to high glucose that is
associated with a decrease in the binding activity of Pdx1
(23). These findings document the importance of Pdx1-
binding activity in regulating pancreatic
-cell expression of the
insulin gene.
For Pdx1 to exert its transcriptional activity, it must translocate
from the cytoplasm to the nucleus. This process involves intracellular
signaling through either phosphatidylinositol 3-kinase (PI 3-kinase) or
stress-activated protein kinase (SAPK)2/p38 pathways (8, 20, 37,
51). The participation of either pathway leads to
phosphorylation of Pdx1, and then Pdx1 enters the nucleus. Stimulation
of nuclear translocation of Pdx1 by either glucose or insulin is
inhibited by either wortmanin, a PI 3-kinase inhibitor, or SB-203580, a
SAPK2 inhibitor. These findings suggest the participation of both PI
3-kinase and SAPK2/p38. Furthermore, exposure of recombinant Pdx1
protein to extract from pancreatic -cells enhances its DNA-binding activity. This change is linked to a shift in molecular mass from 31 to
46 kDa (20). Many studies also indicate that the molecular mass of Pdx1 extracted from cultured pancreatic
-cell lines at various conditions is estimated to be ~46 kDa (37, 42, 47, 48). In addition, it is also reported that there is a
tissue-specific phosphorylation pattern of Pdx1 between pancreatic
endocrine and duct cells (11). However, it is unlikely
that phosphorylation of Pdx1 is responsible for the 15-kDa difference.
Thus we speculate that other posttranslational modification may be
attributed to the increase in molecular mass of Pdx1 and regulate its function.
The marked change in molecular mass of Pdx1 led us to suspect that the
addition of ubiquitin (3, 12) or ubiquitin-like protein to
Pdx1 could account for the change in molecular mass and alter its
function. The small ubiquitin-related modifier 1 (SUMO-1) is a
candidate because it is known to conjugate with target proteins,
including numerous transcription factors (i.e., IB
and p53; see
Refs. 5, 9, 29,
39), and regulates protein functions (25, 30, 43,
50, 53). For example, phosphorylation of Ran-GTPase-activating
protein (RanGAP) permits the addition of SUMO-1 to the protein, which
in turn raises the molecular mass of RanGAP by 20 kDa. This
modification promotes RanGAP translocation from the cytoplasm to the
nucleus (21, 22, 24, 41). Thus, in the present study, we
examined a possibility that the addition of SUMO-1 to Pdx1 causes a
shift of the molecular mass from 31 to 46 kDa to facilitate the
localization and function of Pdx1.
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MATERIAL AND METHODS |
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Plasmids, SUMO-1 RNA interference, recombinant adenoviruses, and
recombinant Pdx1 protein preparation.
The complete coding sequence of mouse Pdx1 was inserted separately in
the plasmid vector pcDNA3.1 (Invitrogen, Carlsbad, CA) to yield
pcDNA-Pdx1 according to a procedure previously described (49). For the construction of the histidine (His)-tagged
SUMO-1 expression vector, the full sequence of human SUMO-1 cDNA was amplified by PCR with specific primers (sense primer
5'-ATGCATCACCATCACCATCACTCTGACCAGGAGGCA, antisense primer
5'-TTTCAAAGAGATGGGGTGCC) and subcloned into EcoRI (TAKARA, Shiga, Japan) sites of the pcDNA3.1 vector. The 374-bp (362
to 12) fragment of the rat insulin-1 promoter gene was cloned with the
primers 5'-ACGCGTACCAGGTCCCCAACAACTGC (sense primer) and
5'-CTCGAGTTAGGGTTGGGAGTTACTGG (antisense primer) using genomic DNA as a
template and subcloned into MluI and XhoI
(TAKARA) sites of pGL3-basic luciferase vector (Promega, Madison, WI)
to yield pINS-Luc.
Cell culture and transfection of expression vector or SUMO-iRNAs.
COS-7 cells (kidney fibroblast cell line from African green monkey,
passages 20-25), HEK-293 cells (kidney epithelial cell line from
Homo sapiens), and -TC-6 cells (insulinoma cell line from
mouse) were purchased from American Type Culture Collection (Rockville,
MD) and cultured continuously in DMEM supplemented with 10%
heat-inactivated FBS, 25 mM D-glucose, 44.0 mM
NaHCO3, 0.1 g/l streptomycin, and 105 U/l
penicillin G at 37°C under a humidified atmosphere of 95% air-5%
CO2.
Treatment of proteasome inhibitor lactacystin. First, COS-7 cells were transfected with SUMO-iRNAs for 48 h, and then the cells were transduced with Ad-Pdx1 and incubated for 48 h with 10 µM lactacystin (Calbiochem-Novabiochem, San Diego, CA) diluted in DMSO (Sigma-Aldrich, St. Louis, MO), which was a proteasome inhibitor. The control cells were incubated with only DMSO for 48 h. Medium containing DMSO or lactacystin was changed every 12 h.
RNA extraction and Northern blot analysis. Total RNA was extracted from the cells using a procedure described previously (52). Aliquots (10 µg) of each sample of total RNA were used for Northern blot analysis. The transferred blots were hybridized with a 32P-labeled fragment of Pdx1 cDNAs. The signal arising from 18S ribosomal mRNA served as the internal control to evaluate differences among various samples.
Preparation of nuclear extracts and Western blot analysis. Preparation of nuclei and cytosolic proteins was performed as described previously (13). Briefly, cytosolic extracts were collected in hypotonic HEPES buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, and 1 mM dithiothreitol) containing protease inhibitors and Nonidet P-40 (NP-40, 0.5%) for 20 min at 4°C. Nuclear extracts were collected with hypertonic HEPES buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol) and agitated for 30 min at 4°C. Next, both extracts were immunoprecipitated with mouse anti-SUMO-1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-His monoclonal antibody (Qiagen), or rabbit anti-SUMO-3 polyclonal antibody (Zymed Laboratories, South San Francisco, CA). Whole cell lysates were extracted with radioimmune precipitation assay buffer (10 mM Tris · HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 1% sodium deoxycholate) and used for Western blot analysis. Cell extracts were incubated for 30 min with 10 units of potato acid phosphatase (Sigma-Aldrich) in 45 mM citrate buffer (pH 4.8).
Western blot analysis was performed according to a procedure described previously (18). Briefly, 10 µg of cellular extracts were separated by the standard SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Specific antibodies against Pdx1 (49) or SUMO-1 protein were used to probe the blots.Immunohistochemical analysis and luciferase reporter assay. The cells were stained as previously described (32). In brief, the cells were fixed for 2 h with 4% paraformaldehyde and washed for 4 days with 0.1 mol/l PBS containing 0.3% Triton X-100 (PBST). For immunofluorescence double staining, the fixed cells were incubated with a mixture of antibodies against Pdx1 and SUMO-1 diluted to 1:5,000 and 1:1,000 in PBST, respectively. The cells were then incubated with the species-specific secondary antibodies conjugated to either FITC or Texas red. The positive reaction was observed under epifluorescence microscopy (Olympus IX70; Olympus, Osaka, Japan), and the images were taken with a charge-coupled device camera (Cool SNAP/HQ; Nippon Roper, Osaka, Japan). The specificity of the positive staining was confirmed by an immunocytochemical absorption study.
Luciferase reporter assay was performed as previously described (31). Briefly, 2 µg of pINS-Luc andStatistic analysis. Results are given as means ± SE, unless otherwise stated. Scheffé's multiple comparison test was used to determine the significance of any differences among more than two groups, and the unpaired Student's t-test was used to determine the significance of any differences between two groups. P < 0.05 was considered significant.
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RESULTS |
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Posttranslational modification of Pdx1.
To determine the molecular mass of endogenous Pdx1 protein, we isolated
subcellular fractions from -TC-6, a pancreatic
-cell line. The
nuclear and cytoplasmic extracts were investigated by Western blot
analysis using an anti-Pdx1 antibody (Fig.
1A). Endogenous Pdx1 protein
in
-TC-6 cells was found predominantly in the nuclear fraction and
had a molecular mass of 46 kDa. The abundance of Pdx1 was not affected
by the concentration of glucose in medium. Others have suggested
(20) that a shift in the apparent molecular mass of Pdx1
from 31 to 46 kDa was involved in nuclear translocation of the protein
in MIN6 cells, but we could not detect the lower molecular mass form of
Pdx1 in
-TC-6 cells.
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Pdx1 protein modification by covalent attachment of SUMO-1.
We wondered whether the 46-kDa form of Pdx1 might be modified by
SUMO-1. To test this possibility, we immunoprecipitated endogenous Pdx1
from -TC-6 cells and exogenous Pdx1 from COS-7 cells using anti-SUMO-1 antibody. Both nuclear and cytoplasmic extracts from these
cells were analyzed using immunoprecipitation with the Pdx1 antibody
and the anti-mouse SUMO-1 or anti-His-tagged antibodies (Fig. 2,
A-C). Negative controls were
prepared from COS-7 cells transfected with empty vector (Fig. 2,
B and C). As shown in Fig. 2, A and
B, a single Pdx1-reactive band was found in the nuclear fraction with molecular mass of ~46 kDa. In addition, the identical band cross-reacted with anti-His-tagged antibody (Fig. 2C)
in COS-7 cells transfected with pcDNA-Pdx1- plus His-tagged-SUMO-1 expression vector. None of these bands was detected in cells
transfected with empty vector alone (Fig. 2B) or with
His-tagged-SUMO-1 expression vector (Fig. 2C). Furthermore,
because the SUMO family consists of SUMO-1, SUMO-2, and SUMO-3
(19, 53), we also tested whether SUMO-2 and SUMO-3
potentially interacted with Pdx1 by immunoprecipitation methods using
anti-SUMO-3 antibody, which cross-reacts with both SUMO-3 and SUMO-2.
However, we did not observe SUMO-3- and/or SUMO-2-reactive bands by the
immunoprecipitation procedure (data not shown). These findings suggest
that the Pdx1 protein found in the nucleus was modified by covalent
attachment to SUMO-1 in both pancreatic and nonpancreatic cells.
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Effects of SUMO-iRNAs on expression of Pdx1.
Uptake of double-strand RNA by insect cell lines causes knockdown
expression of specific genes, resulting from sequence-specific mRNA
degradation (1, 7, 10), so- called iRNA. On the basis of
the findings above, we knocked down SUMO-1 expression and analyzed its
effect on both function and stability of Pdx1. We used a 21-nucleotide RNA duplex (SUMO-iRNAs) to knock down endogenous expression of the
SUMO-1 gene. The COS-7 cells pretreated with SUMO-iRNAs were also
infected with an adenovirus (Ad-Pdx1) to equip the cells with the
ability to express Pdx1 (Fig. 3 and 4).
Lysates from these cells were analyzed by
Western blot analysis using both anti-Pdx1 and anti-SUMO-1 antibodies.
As shown in Fig. 3, A and B, SUMO-iRNAs reduced
SUMO-1 expression by 60% of the control, and the expression of Pdx1
protein content was reduced by 80%. In contrast, no change was
observed in -actin levels in the presence or absence of SUMO-iRNAs
(Fig. 3A). Additionally, Northern blot analysis showed that
no change of the cellular content of Pdx1 mRNA was observed between the
presence and absence of SUMO-iRNAs (Fig. 4, A and
B). These findings show that the decreased expression of
SUMO-1 causes a reduction of the Pdx1 protein contents. Because SUMO-1
does not affect Pdx1 mRNA levels, this observation suggests that SUMO-1
binds or conjugates with Pdx1.
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Nuclear colocalization of SUMO-1 and Pdx1.
To investigate whether Pdx1 could exist in the nucleus in the absence
of endogenous SUMO-1 expression, we performed immunohistochemical analysis using both -TC-6 and COS-7 cells. Both Pdx1 (Fig.
5A) and SUMO-1 (Fig.
5B) strongly colocalized in the nucleus of
-TC-6 cells
(Fig. 5C). Because Pdx1 was expressed heavily in COS-7 cells without SUMO-iRNAs (Fig. 5D), we could not discriminate
between the Pdx1 localization of nucleus and cytoplasm. However, SUMO-1 (Fig. 5E) exclusively expressed in the nucleus of COS-7
cells without SUMO-iRNAs. Thus the merged image for Pdx1 and SUMO-1 clearly revealed that both Pdx1 and SUMO-1 colocalized in the nucleus
(Fig. 5F). However, low levels of SUMO-1 expression (Fig. 5H) were found in the nucleus of COS-7 cells in the presence
of SUMO-iRNAs. In those conditions, Pdx1 (Fig. 5G) was
mainly localized in the cytoplasm, with a little expression in the
nucleus. The merged image revealed that both Pdx1 and SUMO-1 weakly
colocalized in the cells (Fig. 5I). These data suggest that
the sumoylation of Pdx1 was needed for Pdx1 to localize in the nucleus.
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Effects of lactacystin (a proteasome inhibitor) on the
stabilization of Pdx1.
Sumoylation is believed to stabilize the adduct by inhibiting
proteasomal degradation of ubiquitinated protein, and the converse, nonsumoylated Pdx1, is degraded in the proteasome. Therefore, we
hypothesized whether nonsumoylated Pdx1 contents increased and were
possibly detected by Western blot analysis when a proteasome inhibitor
blocked the degradation of Pdx1 in the presence of SUMO-iRNAs to
inhibit the formation of the adduct. To test this hypothesis, we
exposed cells transfected with SUMO-iRNAs to 10 µM lactacystin, a
proteasome inhibitor that blocks proteasome activity by targeting the
catalytic -subunit of the proteasome. In the cells transfected with
SUMO-iRNAs alone, SUMO-1 protein expression decreased, and only the
46-kDa form of Pdx1 was detected, with a marked reduction of its
content (Fig. 6A, lane 4, and
6B). However, the level of the
46-kDa form of Pdx1 was not significantly different between the
presence and absence of SUMO-iRNAs by the addition of lactacystin (Fig.
6B). Additionally, the 46-kDa protein band increased in the
presence of lactacystin compared with that in the absence of
lactacystin (Fig. 6A, lanes 3 and 5,
and 6B). As predicted, the cells, which were transfected
with SUMO-iRNAs and exposed to lactacystin, showed the appearance of
Pdx1 with a molecular mass of 34 kDa (Fig. 6A, lane
5). This is likely nonsumoylated Pdx1 protein. These results
suggest that sumoylation of Pdx1 stabilizes this adduct by inhibiting
its degradation by proteasomes.
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Effects of insulin transcriptional activity on Pdx1 sumoylation.
Finally, we tested the ability of sumoylated Pdx1 to stimulate insulin
promoter activity by use of -TC-6 and COS-7 cells. In this study, we
used a luciferase assay comprised of the rat insulin promoter fused to
the reporter to yield pINS-Luc. The activity of insulin gene expression
was decreased significantly in
-TC-6 cells in the presence of
SUMO-iRNAs (Fig. 7A).
Consistently, COS-7 cells transfected with both SUMO-iRNAs and
pcDNA-Pdx1 exhibited significantly lower transcriptional activity of
insulin gene compared with the cells transduced with pcDNA-Pdx1 alone
(Fig. 7B). These results indicate that the modification by
SUMO-1 is important for Pdx1 to exert its stimulatory activity on
insulin gene transcription.
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DISCUSSION |
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In this study, we found that Pdx1 was posttranslationally modified by SUMO-1. The addition of SUMO-1 to Pdx1 accounted for the 46-kDa form of Pdx1. Additionally, sumoylated Pdx1 enabled this form of the protein to localize in the nucleus and also prevented its degradation by proteasomes. Conversely, the nonsumoylated form of Pdx1 could not localize in the nucleus, and this inability to move into this compartment crippled its transcriptional activity on the insulin promoter. These results indicate that SUMO-1 modification is associated with the localization, stability, and transcriptional activity of Pdx1.
SUMO-1 is a 12-kDa protein of the ubiquitin family. The conjugation of
SUMO-1 to proteins may regulate a number of transcription factors.
Previous studies show that SUMO-1 regulates the subcellular localization of IB
(5), stability of I
B
and
p53 (4, 9, 29, 39), and transcriptional activity of the
heat shock transcription factor 1 (14). However, there are
no previous reports on the association of SUMO-1 with Pdx1. Thus we
propose, based on our findings, SUMO-1 conjugation as a
posttranslational modification of Pdx1. A potential mechanism by which
SUMO-1 modification could regulate the Pdx1 localization was studied
using immunoprecipitation (Fig. 2). Our results showed that Pdx1 was
covalently attached to SUMO-1 in the nucleus. However, we could not
conclude that sumoylation was directly needed for the nuclear
localization and stability of Pdx1. In previous studies, a short
consensus sequence, "KXE," has been described as the SUMO-1
acceptor site in most of the known SUMO-1 substrates (14, 22, 29,
53). The "KKEE" amino acid sequence in the nuclear
localization signal (NLS) of Pdx1 was mutated from lysine to arginine.
However, sumoylation was observed in this mutant protein (data not
shown), suggesting that Pdx1 was sumoylated by the different site of
Pdx1 sequence from the classical consensus motif, KXE. Of course, there
is another possibility, that SUMO-1 may regulate the function and
stability of other proteins, which affect the nuclear localization and
stability of Pdx1. Either SUMO-2 or SUMO-3 is speculated as candidate
proteins that would affect the conjugation with Pdx1, since, like
SUMO-1, SUMO-2/3 may play a similar role. SUMO-1 belongs to a family
including SUMO-2 and SUMO-3 (19, 53), two proteins that
are expressed in a wide range of tissues and cells (40).
However, we found no evidence of either protein in association with
Pdx1 in the present study (data not shown). All of these explanations
are speculative; therefore, we need to further evaluate the exact motif
of the sumoylation site of the Pdx1 molecule or the existence of other
proteins that affect the nuclear localization and stability of Pdx1.
Our present study also showed that the 46-kDa form was the predominant
form of Pdx1 present in both -TC-6 and COS-7 cells transfected with
pcDNA-Pdx1. This result is important because Pdx1 cDNA predicts it to
have 283 amino acids with a molecular mass of 31 kDa (Fig.
1B). The possibility that phosphorylation accounted for the
shift from the 31- to the 46-kDa form of Pdx1 was tested by exposing
Pdx1 to phosphatase. The exposure of the phosphatase yielded a Pdx1 of
slightly smaller molecular mass (Fig. 1B). These findings
suggest that the shift of molecular mass of Pdx1 from 31 to 46 kDa was
mainly explained by sumoylation of Pdx1.
Pdx1 is a homeodomain protein that binds to specific sites within the
insulin gene promoter (34, 35, 44). This transcription factor also contains an NLS believed to be necessary for its transport in nucleus. The NLS is located within the homeodomain of Pdx1 and
comprises seven amino acids (RRMKWKK; see Refs. 13 and
28). Nuclear translocation of Pdx1 is believed to be activated by
signaling pathways through either PI 3-kinase or SAPK2/p38 (8,
20, 37, 51). Both pathways stimulate the phosphorylation of Pdx1 protein. However, the RRMKWKK motif does not contain serine, threonine, or tyrosine residues that may serve as potential phosphorylation sites.
Therefore, there may be another site for phosphorylation in Pdx1, and
the phosphorylation of NLS by itself is not necessary for the
translocation of Pdx1 from the cytoplasm to the nucleus (28). Another phosphorylation site, except in NLS, can be
related to the translocation of Pdx1. Nevertheless, previous reports
(37, 42, 47, 48) are consistent with our present findings
that Pdx1 is specifically localized in the nucleus of mouse insulinoma -TC-6 cells without a shift of molecular mass by glucose
concentration (2.5 and 25.0 mM; Fig. 1A), suggesting that
the activation of nuclear Pdx1 translocation is also initiated by
mechanisms other than phosphorylation.
Next, we wondered whether Pdx1 could localize in the nucleus without
the association of SUMO-1. To solve this question, immunocytochemical detection of Pdx1 was performed using SUMO-iRNAs to suppress endogenous SUMO-1 expression. Recently, it has been shown that iRNA can be used in
cultured mammalian cells (1, 7, 10). The delivery of short
interfering RNAs (siRNAs) binds to a nuclease complex and forms an
RNA-induced silencing complex that targets transcripts by base pairing
between one of the siRNA strands and the endogenous mRNA. Here, we
performed this method for efficient in vitro delivery of siRNAs to
COS-7 cells and demonstrated effective and specific inhibition of
SUMO-1 gene expression in the cells. In -TC-6 cells, both Pdx1 and
SUMO-1 expressed strongly in the nucleus and colocalized. Consistently,
the merged image also revealed that both Pdx1 and SUMO-1 colocalized in
the nucleus in COS-7 cells without SUMO-iRNAs. In contrast, in COS-7
cells transfected with SUMO-iRNAs, SUMO-1 was weakly found in the
nucleus, and Pdx1 was mainly found in the cytoplasm (Fig. 5). It
remains unclear whether Pdx1 conjugation with SUMO-1 is precedent for
the nuclear translocation of Pdx1, but our data support the idea that
SUMO-1 modification is needed to localize Pdx1 in the nucleus.
The biological consequences of the two processes are as follows: sumoylation and ubiquitination are quite different. SUMO-1 shares some identity with ubiquitin; both SUMO-1 and ubiquitin use lysine residues for the covalent conjugation to targets, and the steps of enzyme machinery leading to sumoylation and ubiquitination are mechanistically very similar (5, 15, 17, 36). Conjugation of SUMO-1 can increase stability, change subcellular localization, or affect interactions with partner proteins, but polyubiquitination of proteins is known to target selected proteins for proteolysis by the 26S proteasome (4, 9, 25, 29, 30, 38). Thus another question prompted us to ask whether protein degradation by ubiquitination can occur rapidly in Pdx1 protein without SUMO-1 conjugation. To answer this question, we added a proteasome inhibitor, lactacystin, to inhibit proteolysis and SUMO-iRNAs to prevent endogenous SUMO-1 expression. Our results showed that only the 46-kDa form of Pdx1 was weakly detected in COS-7 cells transfected with SUMO-iRNAs alone. Additionally, the 46-kDa protein band from the cell extracts increased in the presence of lactacystin compared with that in the absence of lactacystin. We also found clear appearance of the 34-kDa form of Pdx1 in the presence of both lactacystin and SUMO-iRNAs (Fig. 6). These results suggest that nonsumoylated Pdx1 is degraded rapidly by ubiquitination in proteasome. Furthermore, it may be true that the Pdx1 · SUMO-1 complex is also degraded in proteasome to some extent. The major results of this experiment indicate that the 46-kDa protein band is markedly reduced by the suppression of endogenous SUMO-1 expression and that double Pdx1 bands in molecular mass of 46 and 34 kDa are detected in the presence of both SUMO-iRNAs and lactacystin. Therefore, the 34-kDa band of Pdx1 is identified as a nonsumoylated one, indicating that Pdx1 sumoylation is prevented from degradation in the proteasome.
In summary, we have examined the transcription factor Pdx1 to understand its marked shift in molecular mass from 31 to 46 kDa. Although previous studies suggested that this change might be the result of phosphorylation of the protein, our findings show that the addition of SUMO-1 to Pdx1 is the more likely cause. The covalent attachment of SUMO-1 is associated with nuclear localization of Pdx1. Thereby, it prevents proteasomal degradation and also enables activation transcription of the insulin gene.
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ACKNOWLEDGEMENTS |
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We thank Dr. Norman C. W. Wong (University of Calgary) and Dr. Hitoshi Yasuda (Shiga University of Medical Science) for helpful advice on this project. We thank Dr. Tomoya Terashima (Shiga University of Medical Science) for providing the histidine-tagged SUMO-1 expression vector.
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FOOTNOTES |
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This work was supported, in part, by a Grant-in-Aid for Scientific Research (no. 14570010) from the Ministry of Education, Science, and Culture of Japan.
Address for reprint requests and other correspondence: A. Kashiwagi, Div. of Endocrinology and Metabolism, Dept. of Medicine, Shiga Univ. of Medical Science, Seta, Otsu, Shiga 520-2192 Japan (E-mail: kasiwagi{at}belle.shiga-med.ac.jp).
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.
First published December 17, 2002;10.1152/ajpendo.00390.2002
Received 3 September 2002; accepted in final form 8 December 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Brummelkamp, TR,
Bernards R,
and
Agami R.
A system for stable expression of short interfering RNAs in mammalian cells.
Science
296:
550-553,
2002
2.
Carty, MD,
Lillquist JS,
Peshavaria M,
Stein R,
and
Soeller WC.
Identification of cis- and trans-active factors regulating human islet amyloid polypeptide gene expression in pancreatic beta-cells.
J Biol Chem
272:
11986-11993,
1997
3.
Ciechanover, A.
The ubiquitin-proteasome pathway: on protein death and cell life.
EMBO J
17:
7151-7160,
1998
4.
Desterro, JM,
Rodriguez MS,
and
Hay RT.
SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.
Mol Cell
2:
233-239,
1998[ISI][Medline].
5.
Desterro, JM,
Rodriguez MS,
Kemp GD,
and
Hay RT.
Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.
J Biol Chem
274:
10618-10624,
1999
6.
Egawa, K,
Sharma PM,
Nakashima N,
Huang Y,
Huver E,
Boss GR,
and
Olefsky JM.
Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance.
J Biol Chem
274:
14306-14314,
1999
7.
Elbashir, SM,
Harborth J,
Lendeckel W,
Yalcin A,
Weber K,
and
Tuschl T.
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Nature
411:
494-498,
2001[ISI][Medline].
8.
Elrick, LJ,
and
Docherty K.
Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1.
Diabetes
50:
2244-2252,
2001
9.
Gostissa, M,
Hengstermann A,
Fogal V,
Sandy P,
Schwarz SE,
Scheffner M,
and
DelSal G.
Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1.
EMBO J
18:
6462-6471,
1999
10.
Hannon, GJ.
RNA interference.
Nature
418:
244-251,
2002[ISI][Medline].
11.
Heimberg, H,
Bouwens L,
Heremans Y,
Van De Casteele M,
Lefebvre V,
and
Pipeleers D.
Adult human pancreatic duct, and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.
Diabetes
49:
571-579,
2000[Abstract].
12.
Hershko, A,
and
Ciechanover A.
The ubiquitin system.
Annu Rev Biochem
67:
425-479,
1998[ISI][Medline].
13.
Hessabi, B,
Ziegler P,
Schmidt I,
Hessabi C,
and
Walther R.
The nuclear localization signal (NLS) of PDX-1 is part of the homeodomain and represents a novel type of NLS.
Eur J Biochem
263:
170-177,
1999
14.
Hong, Y,
Rogers R,
Matunis MJ,
Mayhew CN,
Goodson M,
Park-Sarge OK,
and
Sarge KD.
Regulation of heat shock transcription factor 1 by stress-induced SUMO-1 modification.
J Biol Chem
276:
40263-40267,
2001
15.
Johnson, ES,
Schwienhorst I,
Dohmen RJ,
and
Blobel G.
The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.
EMBO J
16:
5509-5519,
1997
16.
Jonsson, J,
Carlsson L,
Edlund T,
and
Edlund H.
Insulin-promoter-factor 1 is required for pancreas development in mice.
Nature
371:
606-609,
1994[ISI][Medline].
17.
Kamitani, T,
Nguyen HP,
and
Yeh ET.
Preferential modification of nuclear proteins by a novel ubiquitin-like molecule.
J Biol Chem
272:
14001-14004,
1997
18.
Kojima, H,
Nakamura T,
Fujita Y,
Kishi A,
Fujimiya M,
Yamada S,
Kudo M,
Nishio Y,
Maegawa H,
Haneda M,
Yasuda H,
Kojima I,
Seno M,
Wong NC,
Kikkawa R,
and
Kashiwagi A.
Combined expression of pancreatic duodenal homeobox 1, and islet factor 1 induces immature enterocytes to produce insulin.
Diabetes
51:
1398-1408,
2002
19.
Lapenta, V,
Chiurazzi P,
van der Spek P,
Pizzuti A,
Hanaoka F,
and
Brahe C.
SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter, and defines a novel gene family.
Genomics
40:
362-366,
1997[ISI][Medline].
20.
Macfarlane, WM,
McKinnon CM,
Felton-Edkins ZA,
Cragg H,
James RF,
and
Docherty K.
Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells.
J Biol Chem
274:
1011-1016,
1999
21.
Mahajan, R,
Delphin C,
Guan T,
Gerace L,
and
Melchior F.
A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.
Cell
88:
97-107,
1997[ISI][Medline].
22.
Mahajan, R,
Gerace L,
and
Melchior F.
Molecular characterization of the SUMO-1 modification of RanGAP1, and its role in nuclear envelope association.
J Cell Biol
140:
259-270,
1998
23.
Matsuoka, T,
Kajimoto Y,
Watada H,
Kaneto H,
Kishimoto M,
Umayahara Y,
Fujitani Y,
Kamada T,
Kawamori R,
and
Yamasaki Y.
Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells.
J Clin Invest
99:
144-150,
1997
24.
Matunis, MJ,
Coutavas E,
and
Blobel G.
A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.
J Cell Biol
135:
1457-1470,
1996[Abstract].
25.
Melchior, F.
SUMO-nonclassical ubiquitin.
Annu Rev Cell Dev Biol
16:
591-626,
2000[ISI][Medline].
26.
Melloul, D,
Marshak S,
and
Cerasi E.
Regulation of insulin gene transcription.
Diabetologia
45:
309-326,
2002[ISI][Medline].
27.
Miller, CP,
McGehee RE, Jr,
and
Habener JF.
IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.
EMBO J
13:
1145-1156,
1994[Abstract].
28.
Moede, T,
Leibiger B,
Pour HG,
Berggren P,
and
Leibiger IB.
Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1.
FEBS Lett
461:
229-234,
1999[ISI][Medline].
29.
Muller, S,
Berger M,
Lehembre F,
Seeler JS,
Haupt Y,
and
Dejean A.
c-Jun and p53 activity is modulated by SUMO-1 modification.
J Biol Chem
275:
13321-13329,
2000
30.
Muller, S,
Hoege C,
Pyrowolakis G,
and
Jentsch S.
SUMO, ubiquitin's mysterious cousin.
Nat Rev Mol Cell Biol
2:
202-210,
2001[ISI][Medline].
31.
Nakamura, T,
Fox-Robichaud A,
Kikkawa R,
Kashiwagi A,
Kojima H,
Fujimiya M,
and
Wong NC.
Transcription factors and age-related decline in apolipoprotein A-I expression.
J Lipid Res
40:
1709-1718,
1999
32.
Nakamura, T,
Kishi A,
Nishio Y,
Maegawa H,
Egawa K,
Wong NC,
Kojima H,
Fujimiya M,
Arai R,
Kashiwagi A,
and
Kikkawa R.
Insulin production in a neuroectodermal tumor that expresses islet factor-1, but not pancreatic-duodenal homeobox 1.
J Clin Endocrinol Metab
86:
1795-1800,
2001
33.
Offield, MF,
Jetton TL,
Labosky PA,
Ray M,
Stein RW,
Magnuson MA,
Hogan BL,
and
Wright CV.
PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.
Development
122:
983-995,
1996
34.
Ohlsson, H,
Karlsson K,
and
Edlund T.
IPF1, a homeodomain-containing transactivator of the insulin gene.
EMBO J
12:
4251-4259,
1993[Abstract].
35.
Petersen, HV,
Serup P,
Leonard J,
Michelsen BK,
and
Madsen OD.
Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.
Proc Natl Acad Sci USA
91:
10465-10469,
1994
36.
Pickart, CM.
Targeting of substrates to the 26S proteasome.
FASEB J
11:
1055-1066,
1997
37.
Rafiq, I,
da Silva Xavier G,
Hooper S,
and
Rutter GA.
Glucose-stimulated preproinsulin gene expression, and nuclear trans-location of pancreatic duodenum homeobox-1 require activation of phosphatidylinositol 3-kinase but not p38 MAPK/SAPK2.
J Biol Chem
275:
15977-15984,
2000
38.
Rodriguez, MS,
Dargemont C,
and
Hay RT.
SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting.
J Biol Chem
276:
12654-12659,
2001
39.
Rodriguez, MS,
Desterro JM,
Lain S,
Midgley CA,
Lane DP,
and
Hay RT.
SUMO-1 modification activates the transcriptional response of p53.
EMBO J
18:
6455-6461,
1999
40.
Saitoh, H,
and
Hinchey J.
Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3.
J Biol Chem
275:
6252-6258,
2000
41.
Saitoh, H,
Pu R,
Cavenagh M,
and
Dasso M.
RanBP2 associates with Ubc9p and a modified form of RanGAP1.
Proc Natl Acad Sci USA
94:
3736-3741,
1997
42.
Sayo, Y,
Hosokawa H,
Imachi H,
Murao K,
Sato M,
Wong NC,
Ishida T,
and
Takahara J.
Transforming growth factor beta induction of insulin gene expression is mediated by pancreatic and duodenal homeobox gene-1 in rat insulinoma cells.
Eur J Biochem
267:
971-978,
2000
43.
Seeler, JS,
and
Dejean A.
SUMO: of branched proteins and nuclear bodies.
Oncogene
20:
7243-7249,
2001[ISI][Medline].
44.
Serup, P,
Petersen HV,
Pedersen EE,
Edlund H,
Leonard J,
Petersen JS,
Larsson LI,
and
Madsen OD.
The homeodomain protein IPF-1/STF-1 is expressed in a subset of islet cells and promotes rat insulin 1 gene expression dependent on an intact E1 helix-loop-helix factor binding site.
Biochem J
310:
997-1003,
1995[ISI][Medline].
45.
Stoffers, DA,
Zinkin NT,
Stanojevic V,
Clarke WL,
and
Habener JF.
Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence.
Nat Genet
15:
106-110,
1997[ISI][Medline].
46.
Waeber, G,
Thompson N,
Nicod P,
and
Bonny C.
Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.
Mol Endocrinol
10:
1327-1334,
1996[Abstract].
47.
Wang, X,
Cahill CM,
Pineyro MA,
Zhou J,
Doyle ME,
and
Egan JM.
Glucagon-like peptide-1 regulates the beta cell transcription factor, PDX-1, in insulinoma cells.
Endocrinology
140:
4904-4907,
1999
48.
Wang, X,
Zhou J,
Doyle ME,
and
Egan JM.
Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic beta-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism.
Endocrinology
142:
1820-1827,
2001
49.
Watada, H,
Kajimoto Y,
Miyagawa J,
Hanafusa T,
Hamaguchi K,
Matsuoka T,
Yamamoto K,
Matsuzawa Y,
Kawamori R,
and
Yamasaki Y.
PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin.
Diabetes
45:
1826-1831,
1996[Abstract].
50.
Wilson, VG,
and
Rangasamy D.
Intracellular targeting of proteins by sumoylation.
Exp Cell Res
271:
57-65,
2001[ISI][Medline].
51.
Wu, H,
MacFarlane WM,
Tadayyon M,
Arch JR,
James RF,
and
Docherty K.
Insulin stimulates pancreatic-duodenal homoeobox factor-1 (PDX1) DNA-binding activity and insulin promoter activity in pancreatic beta cells.
Biochem J
344:
813-818,
1999[ISI][Medline].
52.
Yamada, S,
Kojima H,
Fujimiya M,
Nakamura T,
Kashiwagi A,
and
Kikkawa R.
Differentiation of immature enterocytes into enteroendocrine cells by Pdx1 overexpression.
Am J Physiol Gastrointest Liver Physiol
281:
G229-G236,
2001
53.
Yeh, ET,
Gong L,
and
Kamitani T.
Ubiquitin-like proteins: new wines in new bottles.
Gene
248:
1-14,
2000[ISI][Medline].