The DNA Binding Activity of the RIPE3b1 Transcription Factor of Insulin Appears to Be Influenced by Tyrosine Phosphorylation*

Taka-aki Matsuoka, Li Zhao, and Roland SteinDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The RIPE3b1 DNA binding factor plays a critical role in pancreatic islet beta  cell-specific and glucose-regulated transcription of the insulin gene. Recently it was shown that RIPE3b1 binding activity in beta  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 beta  cells. We found that RIPE3b1 binding was inhibited upon incubating beta  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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 beta  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 beta  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 beta  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 beta  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 beta  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
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INTRODUCTION
MATERIALS AND METHODS
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Cell Culture and Nuclear Extract Preparation-- Monolayer cultures of beta 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. beta 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 beta  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 beta 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
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INTRODUCTION
MATERIALS AND METHODS
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A beta  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 beta  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 beta  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 beta  cell lines (i.e. HIT T-15 and beta TC-3; data not shown). Collectively, the data suggested that a beta  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.

A beta  Cell Tyrosine Phosphatase(s) Inhibits RIPE3b1 Binding Activity-- To determine whether RIPE3b1 binding was reduced by a beta  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 beta  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.

The 46-kDa DNA Binding Subunit of RIPE3b1 Is Tyrosine-phosphorylated-- Having established that a tyrosine phosphatase(s) in beta  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 beta 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 beta 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 beta  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 (alpha -pTyr) antibody immunoprecipitation procedure. The results shown in B and C are representative of an experiment repeated on five separate occasions. B, beta 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 beta 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 beta 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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ABSTRACT
INTRODUCTION
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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 beta  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 beta  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 beta  cell. The marked abnormalities in glucose homeostasis and cell function observed upon removing the insulin receptor selectively from islet beta  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 beta  cells by acting upon RIPE3b1.

RIPE3b1 binding to the insulin C1 element was dramatically reduced in beta  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 beta  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 beta  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 beta  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 beta  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 beta  cell lines and not within a variety of endocrine (islet alpha , 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by Grant DK 42502 from the National Institutes of Health (to R. S.), by a grant from the Japan Society for the Promotion of Science for Young Scientists (to T. M.), and by the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service Grant P60 DK20593 from the National Institutes of Health).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.

Dagger To whom correspondence should be addressed. Tel.: 615-322-7026; Fax: 615-322-7236; E-mail: Roland.Stein@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M010321200

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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