From the Department of Molecular Physiology and Biophysics,
Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, November 14, 2000, and in revised form, April 2, 2001
The RIPE3b1 DNA binding factor plays a critical
role in pancreatic islet
cell-specific and glucose-regulated
transcription of the insulin gene. Recently it was shown that RIPE3b1
binding activity in
cell nuclear extracts is reduced by treatment
with either calf intestinal alkaline phosphatase (CIAP) or a
brain-enriched phosphatase preparation (BPP) (Zhao, L., Cissell,
M. A., Henderson, E., Colbran, R., and Stein, R. (2000)
J. Biol. Chem. 275, 10532-10537). Evidence is
presented here suggesting that a tyrosine phosphatase(s) influences the
ability of RIPE3b1 to bind to the insulin C1 element in
cells. We
found that RIPE3b1 binding was inhibited upon incubating
cell
nuclear extracts at 30 °C. In contrast, PDX-1 and MLTF-1 transcription factor binding activity was unaffected under these conditions. The loss in RIPE3b1 binding activity was prevented by
inhibitors of tyrosine phosphatases (sodium orthovanadate and sodium molybdate) but not by inhibitors of serine/threonine
phosphatases (sodium fluoride, okadaic acid, and microcystin
LR). CIAP- and BPP-catalyzed inhibition of RIPE3b1 binding was
also blocked by these tyrosine phosphatase inhibitors. Collectively,
the data suggested that removal of a tyrosine(s) within RIPE3b1
prevented activator binding to insulin C1 control element sequences.
The presence of a key phosphorylated tyrosine(s) within this
transcription factor was further supported by the ability of the 4G10
anti-phosphotyrosine monoclonal antibody to immunoprecipitate RIPE3b1
DNA binding activity. We discuss how tyrosine phosphorylation, a very
rare and highly significant regulatory modification, may control
RIPE3b1 activator function.
 |
INTRODUCTION |
Insulin is a polypeptide hormone that plays a critical role in
glucose homeostasis by stimulating the uptake of glucose into cells.
Expression of insulin in adults is limited to pancreatic islet
cells primarily because of the recognition, by specific positive-acting
transcription factors, of its enhancer region, which is located between
nucleotides
340 and
91 relative to the insulin gene transcription
start site (1-3). Selective activation from this region is
predominantly mediated by the C2 (
317 to
311 base pairs) (2), A3
(
205 to
189 base pairs) (4-6), C1 (
115 to
107 base pairs)
(7-9), and E elements (
100 to
91 base pairs) (4, 7, 10).
(These insulin cis-elements are labeled in accordance with
the nomenclature proposed by German et al. (11).) The
factors that act at A3, C1, and E also control glucose-inducible
transcription (5, 8, 12-15), the primary metabolic regulator of
insulin gene expression in vivo.
The C2 (i.e. PAX6 (2)) and A3 (i.e. PDX-1
(6, 16, 17)) activators are islet
cell-enriched homeodomain
proteins, whereas a basic helix-loop-helix-containing protein controls
E element stimulation (i.e. BETA2 (18)). Strikingly, these
proteins regulate gene expression within islet cell types and during
pancreogenesis. Thus, an inactivating mutation in the pdx-1
locus affects a very early development step that prevents both exocrine
and endocrine pancreas formation (19, 20), whereas BETA2 (18) and PAX6 (21) null mutants affect later, but distinct, stages of endocrine islet
cell formation. Moreover, the ability of islet
cells to produce
insulin in sufficient amounts to meet the needs of the body is
compromised in type 2 diabetes mellitus patients with an inactivating
mutation within one allele of the BETA2 (22) and
pdx-1 genes (23, 24). Collectively, these results
established a central role for each of the isolated insulin gene
transcription factors in islet cell development and function.
In contrast to the other primary control elements of the insulin gene,
the C1 element activator, RIPE3b1 (7-9), has not been isolated.
RIPE3b1 DNA binding is mediated by a
cell-enriched protein(s) of
~46 kDa, whose activity is reduced by either calf intestinal alkaline
phosphatase (CIAP)1 or a
brain-enriched phosphatase preparation (BPP) treatment (9). These
results suggested that RIPE3b1 binding is regulated by phosphorylation, a posttranslational modification mechanism also utilized in controlling PDX-1 activation (25, 26). In an effort to more fully understand the role that phosphorylation plays in RIPE3b1 function, we have characterized the phosphatase activity that affects RIPE3b1
binding within the
cell. Our findings strongly suggest the RIPE3b1
activation is influenced by phosphorylation of a tyrosine residue(s)
within the 46-kDa DNA-binding protein.
 |
MATERIALS AND METHODS |
Cell Culture and Nuclear Extract Preparation--
Monolayer
cultures of
TC-3 (27) and MIN6 (28) cells were grown under
conditions described previously. The MIN6 cells were between passages
20 and 29, and insulin transcription and secretion were
glucose-responsive. Human islets were provided by the Juvenile Diabetes
Foundation International Human Islet Distribution Program at Washington
University and were cultured in Connaught Medical Research
Laboratories medium (Life Technologies, Inc.) with 10%
heat-inactivated fetal bovine serum.
TC-3, MIN6, and human islet
nuclear extracts were prepared by the mini-extract procedure described
by Schreiber et al. (29) that involves disrupting the cell
membrane in Nonidet P-40 (Fluka) and resuspending the centrifuged
nuclear pellet in a buffer containing 20 mM HEPES (pH 7.9),
0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and 1 mM
phenylmethylsulfonyl fluoride. Nuclei from MIN6 cells were also
isolated after sucrose gradient centrifugation using the conditions
described by Hallberg (30) and resuspended in the buffer described
above. The extract prepared from sucrose gradient-isolated nuclei was
only used in the experiment described in Fig. 1A. Nuclear
extract (5 µg) was incubated in phosphatase buffer (20 mM
Tris-HCl (pH 7.0), 1 mM DTT, 0.1 mM EGTA, 2 mM MgCl2, 1× protease inhibitor mixture
solution (Roche Molecular Biochemicals)) at 4 or 30 °C for the
indicated period of time. The MIN6 nuclear extract was incubated at
30 °C in phosphatase buffer with CIAP (0.25 µl/reaction (10-µl
reaction mixture volume); 20 units/µl (Promega)) or BPP catalytic
subunit mixture (1 µl/reaction (10 µl), 360 fmol/min using glycogen
phosphorylase (31)). The brain protein phosphatase catalytic
subunit-enriched preparation was prepared by ethanol precipitation of a
rat brain soluble extract (31).
Electrophoretic Mobility Shift Assays--
Nuclear protein
extract (5 µg) was preincubated in mobility shift buffer (10 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM DTT, 10% (v/v) glycerol, 1 µg of poly(dI-dC)) for 15 min at 4 °C (total volume, 50 µl). The binding reaction was
initiated by addition of a double-stranded 32P-labeled
probe (1 ng, 1 × 105 cpm) containing C1
(
126TGGAAACTGCAGCTTCAGCCCCTCTG
101)
and A3 (
214CCTCTTAAGACTCTAATTACCCT
192)
element sequences from the rat insulin II gene and an adenovirus-5 major late transcription factor (MLTF) binding site
(5'-GGTGTAGGCCACGTGACCGGGTGT-3'). The conditions for the insulin C1
competition analyses were the same, except that the wild type,
108/
107 mutant, or
112/
111 mutant (9) was included (in the
amounts detailed in the figure legends) in the reaction. The effect of
the anti-rat N-terminal PDX-1 antisera was analyzed using conditions
described previously (32). Each of the binding reactions was incubated
for 30 min at 4 °C, and the complexes were resolved by
electrophoresis through a 6% nondenaturing polyacrylamide gel using
high ionic strength polyacrylamide gel electrophoresis (PAGE)
conditions (8).
p-Nitrophenyl Phosphate Dephosphorylation--
Twenty µg of
cell nuclear extract was incubated in a reaction mixture containing
10 mM p-nitrophenyl phosphate, 25 mM
sodium acetate (pH 5.0), 20% glycerol, 1.0 mM DTT, and 1.0 mM EDTA (total volume, 100 µl). The relative amount of
para-nitrophenol produced was determined by measuring the
absorbance at 410 nm in 0.2 M NaOH (33), with 100%
phosphatase inhibition activity corresponding to the sample without
nuclear extract.
Anti-phosphotyrosine Antibody Immunoprecipitation--
SDS, to a
final concentration of 0.5% (w/v), was added to
TC-3 nuclear
extract (100 µg) in buffer A (10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 10% glycerol, 1 mM sodium
orthovanadate, and 2 mM DTT). After heating to
65 °C for 10 min (total volume, 30 µl), the SDS concentration was
diluted to 0.05% with buffer A, and 2 µl of anti-Tyr(P)
(4G10, Upstate Biotechnology, Lake Placid, NY) or 2 µl of control
antibody (normal mouse IgG) and 50 µl of protein G-Sepharose beads
were added. The mixture was incubated overnight at 4 °C. The
immunoprecipitate was washed three times with buffer A containing 150 mM NaCl and 1% Triton X-100 and resuspended in 1×
SDS-PAGE loading buffer. The immunoprecipitated proteins were separated
on a 10% SDS-polyacrylamide gel and transferred onto an Immobilon
polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), and
C1 binding activity in the 44-47-kDa range fraction was eluted using
the conditions described previously (9). Phosphotyrosine (2 mM) and phenylphosphate (10 mM) were used to
monitor the specificity of the anti-Tyr(P) immunoprecipitation.
 |
RESULTS |
A
Cell Phosphatase(s) Influences RIPE3b1 DNA Binding
Activity--
The activation potentials of a number of transcription
factors are modulated by their phosphorylation status through effects on such processes as nuclear localization, activation domain activity, and DNA binding (34). For example, phosphorylation of PDX-1 in
glucose-induced
cells appears to stimulate its DNA binding activity
(25, 26, 35). In addition, RIPE3b1 binding activity is reduced by CIAP
and BPP treatment, suggesting that activation of this insulin gene
transcription factor is also mediated by phosphorylation, specifically
of the 46-kDa DNA binding subunit (9).
To determine whether an endogenous protein phosphatase regulated
RIPE3b1 DNA binding activity, MIN6
cell nuclear extracts were
incubated at 30 °C and analyzed for insulin C1 element binding in an
electrophoretic mobility shift assay. Two different procedures were
used to isolate nuclei, one involving Nonidet P-40 and the other
involving sucrose gradient centrifugation. RIPE3b1 activity was reduced
in both nuclear extract preparations during a 30 °C, but not a
4 °C, incubation (Fig. 1A).
The loss in RIPE3b1 activity occurred in a time-dependent
manner, and this effect was only observed on this particular C1 element
complex (Fig. 1, A and B). (The RIPE3b1 complex
was identified by competition analysis with the wild type and
108/
107 binding defective mutant (Fig. 1A).) The loss in
RIPE3b1 binding was prevented by addition of 10 mM sodium
pyrophosphate, a general phosphatase inhibitor (Fig. 1, A
and B). In contrast, binding by PDX-1 (Fig. 1C)
or the generally distributed adenovirus-5 MLTF (Fig. 1D) in
MIN6 nuclear extracts was unaltered by the 30 °C treatment. Because
the DNA binding characteristics of PDX-1 also appear to be regulated by
phosphorylation (25), our inability to detect an endogenous PDX-1
phosphatase most likely indicates that conditions were not optimal to
detect this activity. Importantly, the selective loss in RIPE3b1
binding activity was also observed in nuclear extracts prepared from
human islets (Fig. 2) and other
cell
lines (i.e. HIT T-15 and
TC-3; data not shown).
Collectively, the data suggested that a
cell phosphatase(s) reduced
the DNA binding potential of the RIPE3b1 activator.

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Fig. 1.
RIPE3b1 binding activity is selectively
reduced in MIN6 nuclear extracts incubated at 30 °C. MIN6
nuclear extract (5 µg) prepared from nuclei isolated either with
Nonidet P-40 (NP40) or by sucrose gradient centrifugation
was incubated at 4 or 30 °C in phosphatase buffer with 10 mM NaCl ( ) or with 10 mM NaPPi
(+) for the time period shown. The treated extracts were analyzed for
RIPE3b1 (A), PDX-1 (C), and MLTF (D)
binding using the electrophoretic mobility shift assay. The binding
specificity of RIPE3b1 in A was evaluated using a 10-fold
excess of unlabeled wild type (WT) and 108/ 107 mutant
competitor. The arrow denotes the RIPE3b1 complex. The
amount of the RIPE3b1 (B), PDX-1 (C), and MLTF
(D) binding complex was determined by densitometric
scanning, and the effect of the 30 °C incubation is presented
relative to the 4 °C incubation (t = 0) ± S.E.
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Fig. 2.
Human islet nuclear extracts contain a
RIPE3b1 phosphatase. Human islet nuclear extracts were incubated
for 30 min at 4 or 30 °C in phosphatase buffer in the presence of 10 mM NaCl or NaPPi. RIPE3b1 (A) and
PDX-1 (B) binding was measured using the electrophoretic
mobility shift assay. The RIPE3b1 complex was detected by competition
analysis with the wild type and 108/ 107 mutant C1 oligonucleotide
(not shown); the PDX-1 complex was identified by supershift
(SS) analysis with PDX-1 antiserum. The RIPE3b1 and PDX-1
complexes are labeled.
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A
Cell Tyrosine Phosphatase(s) Inhibits RIPE3b1 Binding
Activity--
To determine whether RIPE3b1 binding was reduced by a
cell serine/threonine or tyrosine phosphatase, inhibitors of each class were included in the MIN6 nuclear extract incubations. Loss of
RIPE3b1 binding activity was essentially prevented by sodium orthovanadate and sodium molybdate, rather selective inhibitors of
tyrosine phosphatase activity (36-39) (Fig.
3A). In contrast, the RIPE3b1
phosphatase(s) was unaffected by treatment with sodium fluoride, a
general serine/threonine phosphatase inhibitor (33, 38, 40, 41) or by
okadaic acid and microcystin LR, which selectively reduce PP1 and PP2A
serine/threonine phosphatase activity (Fig. 3A). The same
regulatory pattern was found when total tyrosine phosphatase activity
in the MIN6 nuclear extract was measured in the presence of these
inhibitors (Fig. 3B). Activators of the PP2B and PP2C
serine/threonine phosphatases, like calcium, calmodulin, and magnesium,
also had no effect on the RIPE3b1 phosphatase(s) activity in MIN6
nuclear extracts (data not shown). In addition, CIAP- (Fig.
3C) and BPP-mediated (Fig. 3D) inhibition of
RIPE3b1 binding was also reduced by the tyrosine phosphatase
inhibitors. However, unlike that observed with MIN6 nuclear extracts
alone, the BPP-catalyzed reduction in RIPE3b1 binding was sensitive to okadaic acid and microcystin LR (Fig. 3D). These findings
suggested that tyrosine phosphorylation of RIPE3b1 played an essential
role in controlling transcriptional activation by this insulin gene transcription factor.

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Fig. 3.
Effect of serine/threonine and tyrosine
phosphatase inhibitors on the RIPE3b1 phosphatase(s) in MIN6 nuclear
extracts. MIN6 nuclear extracts were incubated with general
(NaPPi) or specific tyrosine
(Na3VO4, Na2MoO4) and
serine/threonine (NaF, okadaic acid (OA), microcystin LR
(MCLR)) phosphatase inhibitors at 30 °C for 5 (C, D) or 20 (A, B) min.
The effect of each inhibitor is presented relative to the 10 mM NaCl control ± S.E. (4 °C (A,
C, D); 30 °C (B)). A,
RIPE3b1 binding in the treated extracts was analyzed by gel shift
analysis; B, total tyrosine dephosphorylation activity was
determined using the p-nitrophenyl phosphate assay. CIAP-
(C) and BPP-mediated (D) inhibition of RIPE3b1
binding in MIN6 nuclear extracts was analyzed in the presence of
NaPPi, Na3VO4,
Na2MoO4, okadaic acid, and microcystin
LR.
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The 46-kDa DNA Binding Subunit of RIPE3b1 Is
Tyrosine-phosphorylated--
Having established that a tyrosine
phosphatase(s) in
cells inhibits RIPE3b1 activator binding, we
asked whether there was a tyrosine-phosphorylated protein in the
RIPE3b1 activator complex. To investigate this possibility,
immunoprecipitation studies on
TC-3 cell nuclear extracts were
conducted with the anti-phosphotyrosine 4G10 monoclonal antibody and
normal mouse IgG. These reactions were either conducted with extract
alone or in the presence of extract and competitive inhibitors of
antigen binding (i.e. phosphotyrosine and phenylphosphate).
The immunoprecipitates were collected on protein G-Sepharose beads and
dissociated by addition of SDS (Fig. 4A). These proteins were then
separated by SDS-PAGE and transferred onto an Immobilon polyvinylidene
difluoride membrane, which was cut into slices, and the fractionated
proteins were eluted and renatured. RIPE3b1 element binding was
measured in the 44-47-kDa fraction, which is the only fraction
containing RIPE3b1 DNA binding activity (9). The 4G10 antibody
specifically immunoprecipitated a protein(s) from
TC-3 nuclear
extracts within the roughly 46-kDa fraction that bound to the C1 probe
(Fig. 4B). The immunoprecipitated 46-kDa protein-C1
element complex not only co-migrated with the RIPE3b1 complex detected
in
nuclear extracts (compare lane 1 to 5 in
Fig. 4B) but also bound with the same specificity, as demonstrated by the competition analysis performed with the wild type
and
112/
111 binding defective mutant of C1 (Fig. 4C).
However, the 46-kDa protein was not detected in control
immunoprecipitations with normal mouse IgG or with 4G10 in the presence
of phosphotyrosine or phenylphosphate, thus demonstrating the
specificity of this interaction (Fig. 4B). Collectively,
these results indicate that phosphorylation of a tyrosine(s) in the
46-kDa DNA binding subunit of RIPE3b1 enables this activator to bind
DNA and stimulate transcription.

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Fig. 4.
The DNA binding subunit of RIPE3b1 is
tyrosine-phosphorylated. A, schematic of the
anti-phosphotyrosine ( -pTyr) antibody immunoprecipitation
procedure. The results shown in B and C are
representative of an experiment repeated on five separate occasions.
B, TC-3 nuclear extracts were immunoprecipitated with
anti-phosphotyrosine 4G10 monoclonal antibody, 4G10 plus 2 mM phosphotyrosine, 4G10 plus 10 mM
phenylphosphate, or normal IgG. The immunoprecipitated material was
washed, and the released proteins were electrotransferred from an
SDS-polyacrylamide gel onto an Immobilon polyvinylidene difluoride
membrane. The eluted proteins from the ~44-47-kDa membrane slice
were assayed for C1 binding activity. The untreated TC-3 nuclear
extract was only fractionated by SDS-PAGE/Immobilon chromatography in
lane 5; this complex co-migrates with the RIPE3b1 binding
complex detected in untreated TC-3 nuclear extracts (9). Lane
1, 4G10 alone; lane 2, 4G10 plus 2 mM
phosphotyrosine; lane 3, 4G10 plus 10 mM
phenylphosphate; lane 4, normal IgG; lane 5,
SDS-PAGE/Immobilon only. C, the specificity of
immunoprecipitated 46-kDa protein(s)-C1 probe binding was determined
using a 5-fold molar excess of unlabeled wild type (WT) and
112/ 111 binding mutant competitor to probe. Lane 1,
4G10-immunoprecipitated 46-kDa protein alone ( ); lane 2,
plus wild type; lane 3, plus 112/ 111 mutant.
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|
 |
DISCUSSION |
It is well established that the phosphorylation status of
transcription factors, including those involved in insulin gene expression, can play an essential role in activation. The most common
site of protein phosphorylation is at serine and threonine residues,
and this type of modification has been shown to control the function of
a wide variety of transcription factors (34). For example,
serine/threonine kinase signaling appears to induce PDX-1 activation in
high glucose-treated
cells through effects on both nuclear
translocation and DNA binding (26, 35). In contrast, phosphorylation at
tyrosine residues within transcription factors is found infrequently,
with the only known proteins represented by the signal transducers and
activators of transcription (i.e. STAT 1-6) (42) and
nuclear factor 1-like transcription factor (BEF-1 (43)). However,
because many different types of stimuli that affect gene expression
also lead to the activation of tyrosine protein kinases, it is likely
that other transcription factors will be directly controlled by
tyrosine phosphorylation.
Insulin secretion and transcription from the
cell is
regulated by glucose and secreted insulin. Induced transcription (44) and secretion (45) by insulin is mediated by tyrosine kinase signaling
through the insulin receptor on the
cell. The marked abnormalities
in glucose homeostasis and cell function observed upon removing the
insulin receptor selectively from islet
cells in mice have
established a central role for this signaling pathway in
vivo (46). The data presented here suggest that tyrosine kinase/phosphatase signaling controls insulin gene expression within
islet
cells by acting upon RIPE3b1.
RIPE3b1 binding to the insulin C1 element was dramatically
reduced in
cell nuclear extracts incubated at 30 °C, a process blocked by inclusion of a general phosphatase inhibitor (Figs. 1 and
2). This same pattern of inactivation was found in extracts prepared
from nuclei isolated either with Nonidet P-40 or by sucrose gradient
centrifugation. In contrast, binding of PDX-1 or the generally
distributed adenovirus-5 major late transcription factor was unaffected
under these conditions. Tyrosine phosphatase inhibitors (sodium
orthovanadate and sodium molybdate), but not serine/threonine phosphatase inhibitors (sodium fluoride, okadaic acid, and microcystin LR), prevented the loss in RIPE3b1 DNA binding activity (Fig. 3). These
results indicated that inhibition was caused by dephosphorylation of a
tyrosine(s) within the 46-kDa DNA binding subunit of RIPE3b1 by a
cell nuclear phosphatase(s), a proposal strongly supported by our
ability to specifically immunoprecipitate this insulin C1-binding
protein(s) with the anti-phosphotyrosine 4G10 monoclonal antibody (Fig.
4). However, tyrosine phosphorylation may also influence RIPE3b1
activation by affecting partitioning into the nucleus, as found for the
STAT proteins (42, 47-49), and/or interactions with other
transcriptional regulatory factors. This raises the question of whether
RIPE3b1 activity in glucose-treated
cells is regulated by these
same processes.
The reduction in RIPE3b1 binding by CIAP and BPP was also
prevented by tyrosine phosphatase inhibitors (Fig. 3), consistent with
results associating tyrosine phosphatase activity with the CIAP enzyme
(36, 50) and the brain (51, 52). Interestingly, in contrast to the
cell nuclear extracts, BPP was also less effective at reducing RIPE3b1
binding in the presence of serine/threonine PP1 and PP2A phosphatase
inhibitors (see the OA and MLCR lanes in Fig.
3D). This indicates that RIPE3b1 binding is primarily affected by a tyrosine phosphatase(s) in the
nuclear
protein-enriched preparation but can be affected by both a
serine/threonine and tyrosine phosphatase(s) in a total cellular BPP.
The significance of this observation is under investigation, but it is
noteworthy that STAT 1, 3, and 5 protein activity is also regulated by
both serine/threonine (53-56) and tyrosine phosphatases (48, 49, 56).
Furthermore, the ability of the brain preparation to reduce RIPE3b1
binding activity indicates that this transcription factor may be
present in neuroectodermally derived tissues, a characteristic common
to many transcription factors critical to endocrine islet cell
development and function (i.e. BETA2/NeuroD1 (57), Hlxb9 (58, 59), Isl-1 (60), neurogenin 3 (61), PAX4 (62), PAX6 (21), Nkx2.2
(63), and PDX-1 (19, 20)).
The RIPE3b1 activator appears to be an unidentified
tyrosine-phosphorylated transcription factor, a conclusion based upon the following observations. First, RIPE3b1 binding activity is only
found within islet
cell lines and not within a variety of endocrine
(islet
, exocrine, liver, and kidney) and non-endocrine cell types
(7, 9), unlike the more widely distributed tyrosine-phosphorylated STAT
and BEF-1 transcription factors. Second, the RIPE3b1 DNA-binding protein is much smaller (~46 kDa) than other tyrosine-phosphorylated proteins (STATs, ~90-115 kDa (64); BEF-1, 98 kDa (43)). Furthermore, STAT protein activity is induced by cytokine and growth factor (e.g. epidermal growth factor and platelet-derived growth
factor) treatment (65) and not apparently by glucose (66), whereas RIPE3b1 binding is regulated by glucose levels (8, 13) and not by
epidermal growth factor and platelet-derived growth factor (data not
shown). Based upon this reasoning and results demonstrating the
importance of islet-enriched transcription factors in pancreatic development and islet cell function, we are working toward isolating the cDNA (gene) encoding for the 46-kDa DNA-binding protein of RIPE3b1.
We are grateful to Drs. Roger Colbran, Brian
Wadzinski, and Joe Zhao for assistance in designing and interpreting
many of the experiments described here. Also, we are thankful to Dr.
Miyazaki for providing the MIN6 cells and Dr. Chris Wright for the
PDX-1 antiserum.
The abbreviations used are:
CIAP, calf
intestinal alkaline phosphatase;
BPP, brain-enriched phosphatase
preparation;
DTT, dithiothreitol;
MLTF, major late transcription
factor;
PAGE, polyacrylamide gel electrophoresis;
STAT, signal
transducer and activator of transcription.
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