From the Department of Internal Medicine B, University
Hospital, 1011 Lausanne, Switzerland
JIP-1 is a cytoplasmic inhibitor of the c-Jun
amino-terminal kinase activated pathway recently cloned from a mouse
brain cDNA library. We report herein the expression cloning of a
rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic
-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was
isolated by its ability to bind to GTII, a cis-regulatory
element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino
acid protein, which differs from JIP-1 by the insertion of 47 amino
acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative
helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and
280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells
transfected with an expression vector encoding IB1. Using a
heterologous GAL4 system, we localized an activation domain of IB1
within the first 280 amino acids of the protein. These data demonstrate
that IB1 is a DNA-binding protein related to JIP-1, which is highly
expressed in pancreatic
-cells where it functions as a
transactivator of the GLUT2 gene.
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INTRODUCTION |
In an attempt to identify DNA-binding proteins necessary for
proper
-cell-specific expression of genes in the endocrine pancreas, we initiated the characterization of the promoter of the GLUT2 gene.
GLUT2, a facilitated glucose transporter isoform, is a membrane protein
present in pancreatic
-insulin-secreting cells, the basolateral membrane of intestinal and kidney absorptive cells, in hepatocytes, and
in a subset of neurons (1-3). In several experimental models of
diabetes, GLUT2 expression is dramatically reduced in pancreatic
-cells, and it has been suggested a role for GLUT2 in the
pathogenesis of the disease (4-10). We and others have shown that a
fragment of the GLUT2 promoter displayed glucose responsiveness when
transfected into differentiated insulin-producing cells or into
hepatocytes (11-13). Important cis-regulatory sequences
were identified within this promoter region, including functionally
responsive PDX-1 and cyclic AMP-responsive elements and three
cis sequences named GTI, GTII, and GTIII (13-15). The
minimal promoter region containing GTI, GTII, and GTIII is both
sufficient and necessary to confer pancreatic expression to a reporter
gene in vitro or in vivo in transgenic mice (14,
16). Nuclear proteins specifically expressed in pancreatic
-cells
interact with the GTII sequence (14).
In this report, we describe the expression cloning of a GTII-binding
protein from a pancreatic
-cell cDNA library. The gene encodes a
cDNA abundantly expressed in the pancreatic islets and in the
brain, which was named IB-1 for Islet-Brain 1 (17). A GeneBankTM data
base search with the IB1 cDNA revealed that IB1 is a rat homologue
of the murine cytoplasmic inhibitor of the c-Jun amino-terminal kinase
(JNK)1-activated pathway
termed JIP-1 (18). IB1 differs, however, from JIP-1 by the insertion of
a 47-amino acid region in its carboxyl-terminal part. This insertion
encodes a phosphotyrosine interaction
domain (PID) and a helix-loop-helix motif (HLH).
Furthermore, IB1 is a cytoplasmic and nuclear DNA-binding protein,
which functions as transactivator of the GLUT2 gene.
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MATERIALS AND METHODS |
Construction of an INS-1 cDNA Expression Library and Cloning
of the IB1 cDNA--
An oligo(dT)-primed cDNA was generated
from 10 µg of poly(A)+ RNA obtained from the
differentiated INS-1 insulin-secreting cell line using a cDNA
synthesis kit (Stratagene, La Jolla, CA) according to the
manufacturer's instructions. The cDNAs were cloned into the
EcoRI and XhoI sites of the
Zap Express
expression vector (Stratagene). A total of 2 × 106
colonies were screened by the procedure described by Singh et al. (19) using as probe concatanated GTII oligonucleotides (14). One GTII-interacting positive clone was obtained from the screening and
excised. The resulting cDNA in the pBKS plasmid (pBKS-IB1) was
sequenced in both 5
and 3
orientations.
Cell Lines, Plasmid Constructions, Transient Transfections, and
Luciferase Assays--
The transplantable x-ray induced rat insulinoma
INS-1 cell line was kindly provided by Asfari et al. (20)
and grown as described. The mouse insulin-producing
TC3 cell line
and the kidney-derived COS-7 cell line were cultured as described
previously (21, 22).
The eukaryotic expression vector encoding IB1 was constructed by
inserting the IB1 cDNA in the NheI/XhoI sites
of the CMV-driven plasmid PBKS (Stratagene) to generate the pCMV-IB1
vector. Polymerase chain reaction mutagenesis was used to add a Flag
epitope (Eastman Kodak Co.) in the pCMV-IB1 construct 3
of the
initiating methionine. The
338 bp of the murine GLUT2 promoter (14)
were cloned 5
of a luciferase gene (pGL3Basic vector, Promega,
Madison, WI). For the GAL4 constructs, the IB1 cDNA was introduced into
the pSG147 vector, in frame with the GAL4 DNA-binding domain (aa
1-147) of this vector (23). The luciferase reporter construct used in
the GAL4 system was obtained by linking five copies of the GAL4
DNA-binding sites (5 × GAL4 DNA-binding sites) 5
to the minimal
herpes simplex virus thymidine kinase promoter in the pGL3-TK plasmid
(Promega).
All constructs were transiently transfected using the cationic reagent
DOTAP (Boehringer Mannheim, Mannheim, Germany) as described previously
(14). Luciferase activities were measured according to the protocol of
Brasier et al. (24).
SouthWestern Experiments, in Vitro Transcription and Translation,
RNA, and Northern Blot Analysis--
Nuclear and cytoplasmic extracts
were prepared according to the method of Dent and Latchmann (25). The
SouthWestern experiments were conducted as described (14). The in
vitro translation experiments were performed from the pBKS-IB1
plasmid as template using the coupled transcription-translation kit
(TNT) from Promega and according to the manufacturer's instructions in
the presence of [35S]methionine. The RNA isolation and
Northern blot analysis from rat tissues or cell lines were conducted
exactly as described previously (14). The rat pancreatic islets were
isolated by the method of Gotoh et al. (26).
Preparation of Antisera--
Anti-IB1 antiserum was prepared
using a cDNA fragment encoding the first 280 amino acids of the
protein. This fragment was inserted into the His-tagged pQE-9
expression vector (Qiagen, Basel, Switzerland), expressed, and purified
through a Ni2+-containing column following instructions
from the manufacturer. Purified material was used to elicit polyclonal
antibodies in rabbits. To affinity-purify the antibodies, the
Ni2+-column-purified 1-280 aa of the recombinant protein
were immobilized onto a nitrocellulose membrane, and the rabbit
antiserum (diluted 1:50 in phosphate-buffered saline) was incubated
with this membrane. The membrane was then washed in phosphate-buffered
saline buffer, and the anti-IB1 antibody was eluted by 0.2 M Tris-glycine, pH 2.8, followed by neutralization at pH
~7.5. The preimmune serum was treated in a similar fashion to provide
non-immune control.
Immunohistochemistry--
The immunocytochemistry was performed
essentially as described previously (16). The sections were incubated
for 14 h at 4 °C with the affinity-purified preimmune or immune
anti-IB1 serum (dilution 1/200).
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RESULTS AND DISCUSSION |
Isolation and Sequence Analysis of the IB1 cDNA--
A
DNA binding activity to GTII was shown to be restricted to
insulin-secreting cells (INS-1 and
TC3) (14). A poly(dT)-primed INS-1 cDNA expression library was constructed and screened by the
procedure described by Singh et al. (19) using a
concatanated GTII oligonucleotide probe. One positive clone was
isolated from a primary screen of approximately 2 × 106 phage plaques. The 2,953-bp-long insert encoded a large
open reading frame of 714 amino acids and was termed IB1, for
Islet-Brain 1, as its expression was primarily restricted to these two
tissues, as discussed below (17). A GeneBankTM data base search
revealed that IB1 is a rat homologue of the recently identified JIP-1
protein (18). JIP-1 is a cytoplasmic inhibitor of the JNK-activated pathway, which was cloned from a mouse brain cDNA library using a
two-hybrid system (18). Amino acid and nucleic acid comparison of mouse
JIP-1 and rat IB1 showed that the proteins are almost identical (97%
identity) with the exception of a 47-amino acid addition in the
carboxyl-terminal part of IB1. As depicted in Fig.
1, this 47-amino acid insertion contains
a putative helix-loop-helix domain as well as a PID. PID domains are an
average length of 100-160 amino acids and consist of four conserved
blocks (27, 28). The first block of the putative PID domain of IB1 is
contained in the 47-amino acid insertion and therefore is absent from
the JIP-1 protein. JIP-1 was shown to be a cytoplasmic protein that caused cytoplasmic retention of JNK and that inhibited the
JNK-regulated gene expression (18). As JNK binds in the nuclei to the
transcription factors c-Jun and ATF2, the sequestration of JNK by JIP-1
in the cytoplasm inhibits the JNK signaling pathway. One may speculate that the insertion of the 47 amino acids in the JIP-1 protein, which
creates a HLH and a PID domain, will allow protein-protein interactions, possibly with other members of the tyrosine kinase signaling pathway or with transcription factors.

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Fig. 1.
IB1 differs from JIP-1 by the insertion of 47 amino acids, which contain a HLH and PID domain. A,
schematic diagram of IB1 and JIP1 with the putative motifs
(HLH = helix loop helix; PID = phosphotyrosine interaction domain; NTS = nuclear
translocation signal). B, amino acid sequence comparison of
IB1 with other bHLH proteins (31-34). Shaded amino acids
are conserved in at least five of the sequences represented.
C, sequence alignments of the PID domain of IB1 with several
members of PID-containing proteins (35-39). Shaded amino
acids are conserved in at least five of the sequences represented
(27).
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By computer analysis using the SOPMA algorithm (Self Optimized
Prediction Method of Alignments, CNRS, Lyon, France), two acidic helicoidal structures (aa 31-61 and 114-125) and a proline-rich region (aa 292-366) in the amino-terminal part of IB1 were also predicted, which could act as transactivation domains (29). Putative
nuclear localization signals were also localized at aa 163-190 and
242-270 (30).
IB1 Is Expressed in Pancreatic Insulin-secreting Cells--
IB1,
as JIP-1, was highly expressed in the brain and, to a lower extent, in
the kidney (17, 18). In addition, IB1 was also abundantly expressed in
several insulin-secreting cell lines (INS-1, RIN5F) as well as in
freshly isolated rat pancreatic islets, but not in the liver or in RNA
prepared from whole pancreas, since the pancreatic islets represent a
small proportion of the organ (Fig. 2,
A and B). In pancreatic islets, IB1 expression
was not regulated by increasing the glucose concentration in the
incubation medium from 2.8 to 30 mM (Fig. 2B).
Affinity-purified antibodies detected a 120-kDa protein in nuclear
extracts prepared from
TC3 cells and in crude cellular extracts
prepared from freshly isolated pancreatic islets (Fig. 2, C
and D). This 120-kDa protein comigrated with the product
obtained by in vitro transcription-translation of the IB1
cDNA in the presence of [35S]methionine (data not
shown). We could also detect the IB1 protein in both nuclear and
cytoplasmic extracts obtained from COS-7 cells transiently transfected
with the CMV-driven IB1 cDNA (Fig. 2E).

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Fig. 2.
IB1 is abundantly expressed in
insulin-secreting cells. A, five micrograms of
poly(A)+ RNA prepared from two different insulin-secreting
cell lines, from rat liver, kidney, and whole pancreas, were analyzed
by Northern blotting for IB1 gene expression. B, a total of
5 µg of RNA obtained from isolated rat pancreatic islets incubated in
2.8 or 30 mM glucose for 14 h were analyzed by
Northern blotting, together with rat liver and adipose tissue RNAs.
C, Western blot analysis of TC3 whole cell extracts with
the -IB1 antibody demonstrated the presence of a 120-kDa product
that was not detected with the preimmune serum (CTRL).
D, the IB1 protein is detected in 30 µg of whole cell
extracts obtained from isolated rat pancreatic islets when analyzed by
Western blotting with the -IB1 antibody. E, a plasmid
containing the IB1 cDNA driven by a CMV promoter or its parent
vector was transiently transfected into COS-7 cells and cytoplasmic
(CE) or nuclear (NE) extracts prepared 48 h
after transfection. By Western blot analysis, IB1 is detected in the cytoplasm and the nucleus of the transfected cells.
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To gain further insight into the tissue and cellular localization of
IB1 within the pancreas, immunohistochemistry studies were performed on
mouse islets and
TC3 cells. Affinity-purified antibodies raised
against IB1 detected this factor in the pancreatic islet as well as in
the nuclei and the cytoplasm of
TC3 cells (Fig.
3, A and C). To
confirm the specificity of the anti-IB1 antibodies in
immunocytochemistry, a construct was generated that includes a Flag
epitope located NH2-terminal to the IB1 protein expressed
under the control of a CMV promoter. This construct was transiently
transfected into COS-7 cells, and the translated product was
immunodetected with an anti-Flag antibody subsequently visualized by
fluorescein isothiocyanate staining (Fig. 3E) or with the
anti-IB1 antibody detected using an anti-rabbit Texas Red-labeled
antibody (Fig. 3F). The IB1 protein, in transfected COS-7
cells, was detected with both the anti-Flag and the anti-IB1 antibodies
in the cytoplasm and the nuclei of COS-7 cells.

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Fig. 3.
Immunostaining analysis of IB1 expression.
A, mouse pancreatic sections were immunostained with
affinity-purified -IB1 antibodies, and the detection was performed
using a secondary avidin-biotin-peroxidase complex. IB1 staining is
present in pancreatic islet cells but was absent from the exocrine
cells surrounding the islets. B, preimmune serum control in
TC3 cells. C, TC3 cells immunostained with -IB1
antibodies. D, a plasmid containing a CMV promoter-driving
expression of the IB1 sequence linked to a Flag epitope was transiently
transfected into COS-7 cells (dark field photomicrograph).
E, same cells as in D. The Flag epitope was
detected by indirect immunofluorescence using an anti-Flag antibody and
visualized with a fluorescein-labeled anti-mouse secondary antibody.
F, the same transfected COS-7 cell as in D and
E was immunodetected with the affinity-purified -IB1
antibody and a secondary anti-rabbit Texas Red-labeled antibody.
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The IB1 Protein Binds through Its Carboxyl-terminal Domain to
GTII--
The IB1 cDNA was cloned 3
to a CMV promoter and
transiently transfected into COS-7, a cell line that does not express
endogenous IB1. Crude cellular extracts prepared from these transfected
cells were then analyzed by the SouthWestern technique using the GTII probe. A 120-kDa GTII-binding protein was detected by SouthWestern (Fig. 4), and this size product was
similar to the one obtained by in vitro translation of the
IB1 cDNA in the presence of [35S]methionine and to
the protein detected by anti-IB1 antibodies (Fig. 2). The IB1 cDNA
is therefore translated into a 120-kDa product, which is able to bind
the GTII probe. Constructs were then generated that include only the
amino-terminal part (aa 1-280) or the COOH-terminal part (280-714) of
the protein, and bacterially produced recombinant IB1 proteins were
obtained from these plasmids. The 280-714-aa protein, but not the
1-280-aa protein, was able to bind to the GTII cis sequence
when tested by SouthWestern analysis implying that the carboxyl end of
the protein contains the DNA-binding domain of IB1 (data not
shown).

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Fig. 4.
The DNA binding activity of IB1.
Plasmids containing the IB1 cDNA driven by a CMV promoter
(pCMV-IB1) or its parent vector (pCMV) were
transiently transfected into COS-7 cells, and nuclear extracts (20 µg) of these transfected cells were examined by SouthWestern analysis
with the labeled GTII probe. The 120-kDa expressed protein was detected
only in the transfected cells with the eukaryotic expression vector
containing the IB1cDNA.
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Transcriptional Activation by IB1--
Transcriptional activation
by IB1 was assessed by cotransfection experiments in the
insulin-secreting cell line
TC3, an IB1 expression vector
(pCMV-IB1), and the proximal region of the GLUT2 promoter (
338 bp)
linked to a luciferase reporter gene. Overexpression of IB1
transactivated the GLUT2 promoter 1.6 (±0.1)-fold when compared with a
cotransfection with the expression vector lacking the IB1 cDNA
(PBKS). This effect was absent with the promoterless reporter construct
(pGL3). To avoid possible interference with endogenous IB1 protein
interacting with GTII and/or heterodimerization of IB1 with related
factors, aa 1-268 and 1-714 of IB1 were fused with the GAL4
DNA-binding domain. The GTII-binding sites of the reporter gene were
replaced with GAL4-binding sites linked to a minimal thymidine
kinase-luciferase gene. As shown in Fig.
5, the amino-terminal part of IB1 was
sufficient to confer transactivating functions to this heterologous
GAL4 system (9.0 ± 0.4 and 7.5 ± 1.2 versus
control with the 1-268 and 1-714 GAL4 constructs, respectively).

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Fig. 5.
Transacting functions of IB1. 1-280- or
1-714-aa fragments of the IB1 protein were fused to a heterologous
GAL4-binding domain and tested in transient transfection studies using
as reporter gene multimerized GAL4-binding sites linked to a luciferase
gene.
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In summary, we describe herein the expression cloning, using a
cis-regulatory element of the GLUT2 promoter, of a protein preferentially expressed in the brain and the insulin-secreting cells
named IB1 (17). The rat IB1 amino acid sequence is 97% identical to
the recently described murine JIP-1 cytoplasmic protein (18). JIP-1 and
IB1 differ mainly by the presence of a 47-amino acid insertion in the
carboxyl-terminal part of IB1, which complements a truncated HLH and
PID domain (27, 28). JIP-1 was cloned by its ability to bind to JNK,
implying the existence of protein-protein interactions, while IB1 was
isolated by its ability to bind to a cis-regulatory element
of the GLUT2 promoter, implying DNA-protein interactions. This DNA
binding activity was not suspected for JIP-1, and several lines of
evidence suggest that indeed IB1 is a DNA-binding protein and acts as a
transcriptional factor. First, IB1 was cloned based on its ability to
bind to the GTII cis element. Second, the SouthWestern
experiments could detect IB1 in nuclear extracts of transfected COS-7
cells with the expression vector encoding IB1. Third, immunodetectable
nuclear staining was present in pancreatic islets as well as in
TC3
cells. Fourth, Western blot analysis of these cells could also detect
IB1 in the cytoplasm and in the nucleus.
Further work will be needed to identify other potential partners of the
IB1/JIP-1 proteins, either other transcriptional factors or other
members of the JNK signaling pathway. It remains to be elucidated
whether IB1/JIP-1 plays a differential functional role in the
insulin-secreting cells, in particular in the control of glucose-induced insulin secretion.
We are grateful to Myriam Steinmann for
excellent technical assistance and to Vincent Mooser, Phil Shaw,
Bernard Thorens and Nancy Thompson for critical comments.