©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
GABP Mediates Insulin-increased Prolactin Gene Transcription (*)

(Received for publication, February 21, 1996)

Liaohan Ouyang (§) Kirsten K. Jacob Frederick M. Stanley

From the Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-related transcription factor GABP in nuclear extracts from GH cells. Expression of GABPalpha and GABPbeta1 squelches insulin-increased prolactin gene expression. GABPalpha and GABPbeta1 bind the insulin-response element of the prolactin promoter, and anti-GABPalpha and anti-GABPbeta1 antibodies supershift a species seen with nuclear extracts from GH cells. GABPalpha immunoprecipitated from insulin-treated, P-labeled GH cells was phosphorylated 3-fold more than GABPalpha from control cells. There was no increase in phosphorylation of GABPbeta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABPalpha while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABPalpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation.


INTRODUCTION

The activation of gene transcription by hormones that function through protein-tyrosine kinase receptors is not well understood in comparison with that mediated by other classes of hormones. The receptors for the steroid-thyroid hormones are transcription factors that are activated by hormone binding(1) . G(s) protein-coupled receptors activate gene transcription following hormonal initiation of a cascade ending in the phosphorylation of CREB/ATF transcription factors(2) . Recently, numerous cytokines have been shown to activate transcription via phosphorylation of cytosolic ISGF-3 proteins that then become nuclear localized transcription factors(3) . The responsiveness of genes to each of these classes of hormones is dependent on the presence in the gene of the appropriate DNA sequence to which the activated transcription factor binds. Neither the hormone-responsive DNA element nor the transcription factors activated by protein-tyrosine kinase receptors are known.

Recently, we have identified an insulin-response element in the prolactin promoter that is identical to the binding site for the Ets-related transcription factors(4) . This element also mediates the insulin sensitivity of the thymidine kinase and somatostatin promoters in both HeLa and GH4 cells and confers insulin responsiveness to the mammary tumor virus promoter when it is added to that promoter at -88. Further, the increase in the transcription of these genes in insulin-treated cells was inhibited by expression of a dominant-negative Ets protein(5) . These studies identify the predominant Ets-related protein of GH4 cells, GABPalpha, and suggest that GABP mediates insulin-increased prolactin gene expression. Phosphorylation of GABP by MAP (^1)kinase may regulate its activity.


EXPERIMENTAL PROCEDURES

Materials

[P]dCTP, 3000 Ci/mmol, [P]ATP, 3000 Ci/mmol, and [^14C]chloramphenicol, 50 mCi/mmol, were obtained from ICN Biochemicals Corporation. [P]H(3)PO(4) was from DuPont NEN. All enzymes and linkers were obtained from either New England Biolabs or from Boehringer Mannheim and, unless otherwise indicated, were used under conditions recommended by the suppliers. Oligonucleotides were purchased from Operon. Duplex poly(dIbulletdC) was obtained from Pharmacia Biotech Inc. Antibodies to MAP kinase (anti-Erk-1 and anti-Erk-2), antibody to the DNA binding domain of cEts-1 (pan-Ets), and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies directed against GABPalpha and GABPbeta1 were the generous gift of Dr. S. L. McKnight (Tularik, South San Francisco, CA). Reagents used for gel electrophoresis were purchased from Fisher Scientific. Protein A agarose, acetyl-CoA, and silica gel plates were obtained from Sigma. Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (DMEM) was from Life Technologies, Inc., and iron-supplemented calf serum was obtained from Hyclone Laboratories. Triton X-100, reagents for enhanced chemiluminescence, and BCA reagent were from Pierce. All other reagents were of the highest purity available and were obtained from Sigma, Behring Diagnostics, Bio-Rad, Eastman, Fisher, or Boehringer Mannheim.

Plasmids

The construction of pPrl-CAT plasmids containing -173/+75 of prolactin 5`-flanking DNA was described(6) . The human insulin expression vector, pRT3HIR2, was the gift of Dr. J. Whittaker (Stony Brook, NY). The plasmids CMV-GABPalpha and CMV-GABPbeta were provided by Dr. C. Thompson (Carnegie Institute, Baltimore, MD). The Elk-1 and SRF expression vectors were the gift of Dr. R. Treisman. The GST-GABPalpha expression plasmid was constructed by ligating a blunt-ended BamHI/XhoI fragment from the GABPalpha cDNA (plasmid F27, Dr. S. L. McKnight) into the blunt-ended EcoRI site of pGEX 2T (Pharmacia). The fusion protein consists of the GST protein (26 kDa) and GABPalpha-(76-336) (30 kDa) that contains the three potential MAP kinase phosphorylation sites.

Western Immunoblot Analysis

GH4 cells were harvested after 48 h with or without 1 µg/ml insulin, and nuclei were prepared as described(7) . A nuclear extract was prepared by disrupting the nuclei with 400 mM KCl in a buffer containing 15% glycerol, 25 mM Tris, pH 8, 10 mM beta-mercaptoethanol, 0.5 mM EDTA, and 0.05% Triton X-100. SDS-polyacrylamide gel electrophoresis was performed using 12% gels(8) . The proteins were then blotted to nitrocellulose membranes (Micron Separations) in Towbin's buffer (25 mM Trizma base, 192 mM glycine, and 20% methanol). Immunoblotting using enhanced chemiluminescence was performed as described by the manufacturer (Pierce).

Analysis of Prolactin Promoter Responsiveness Using Transient Transfection

Electroporation experiments and CAT assays were performed as described(9) . GH4 cells were placed for 24 h in DMEM containing 10% hormone-depleted serum (9) and harvested with an EDTA solution, and 20-40 times 10^6 cells were used for each electroporation. Trypan blue exclusion before electroporation ranged from 95 to 99%. The voltage of the electroporation was 1550 V. This gives trypan blue exclusion of 70-80% after electroporation. A Rous sarcoma virus-betaGal expression plasmid was used to control for differences between electroporations as described(9) . The transformed cells were then plated in multiwell dishes (Falcon Plastics) at 5 times 10^6 cells/9-cm^2 tissue culture well in DMEM with 10% hormone-depleted serum. Cells were refed with DMEM with 10% hormone-depleted serum ± insulin at 24 h. After 48 h, the flasks were washed three times with normal saline and frozen. The cells were harvested in hypotonic lysis buffer using a rubber policeman. CAT activity was assayed as described(10) .

Assay of DNA-Protein Binding by Gel Electrophoresis

An oligonucleotide to the prolactin promoter sequence -106/-87 was prepared, purified on polyacrylamide gels, and end-labeled with [P]dCTP. The sequence of this oligonucleotide is 5`-TCTTAATGACGGAAATAGAT-3`. Labeled prolactin 5`-flanking DNA was then used in mobility shift experiments with unlabeled nuclear extracts performed as described(7) . Two µg of nuclear extract were incubated at 25 °C for 30 min with 10,000 cpm (10-20 fmol) of P-labeled Prl -106/-87. The protein-DNA complexes were then analyzed by electrophoresis on a 4% polyacrylamide gel in Tris/acetate/EDTA buffer.

Phosphorylation of GABPalpha by MAP Kinase

GH4 cells were treated for 5 min with 1 µg/ml insulin or were left untreated as controls. They were then washed and frozen at -70 °C. The cells were lysed in a buffer consisting of 50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1.5 mM MgCl(2), 1 mM EGTA, 10% glycerol, 1 mM Na(3)VO(4), 50 mM Na(4)P(2)O(7), 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin. Immunoprecipitations were performed for 90 min at 4 °C in this buffer using 200 µg of protein. Antibody to MAP kinase was obtained from Santa Cruz, and 1 µg was used for each immunoprecipitation. The kinase assay was performed with immunoprecipitated MAP kinase and 0.1 µg of GST protein. The fusion protein consists of the GST protein (26 kDa) and GABPalpha-(76-336) (30 kDa) that contains the three potential MAP kinase phosphorylation sites. The assay was performed in a buffer containing 25 mM HEPES, pH 8.0, 50 µM ATP, and 10 µCi of [P]ATP, 10 mM MgCl(2), and 1 mM dithiothreitol(11) . The supernatant was precipitated with 10% trichloroacetic acid and electrophoresed on a 10% SDS-polyacrylamide gel.

Labeling and Immunoprecipitation of GABPalpha and GABPbeta

GH4 cells in 9-cm^2 tissue culture wells were incubated for 2 h with phosphate-free DMEM containing 10% dialyzed, charcoal-treated calf serum. They were then washed and incubated for 2 h in phosphate-free DMEM containing 10% dialyzed, charcoal-treated calf serum with 0.5 mCi/ml [P]H(3)PO(4). Insulin was then added and the incubation was continued for 1 h. The cells were rapidly chilled, washed 3 times with ice-cold saline, and frozen at -70 °C. The cells were then processed and immunoprecipitated as described above.


RESULTS AND DISCUSSION

Immunoblot analysis of GH4 cell nuclear extracts was performed to determine which of the Ets-related transcription factors might mediate the effects of insulin on prolactin gene expression (Fig. 1). A pan-Ets antibody was used to visualize Ets-related proteins in GH4 cell nuclear extracts. This antibody was raised against the conserved DNA binding domain of Ets-1, and it demonstrates broad cross-reactivity with Ets family proteins. One band of approximately 51 kDa was visible using either 1 or 3 µg of nuclear extract. This is the same size as the previously identified Ets-related protein GABPalpha. GABPalpha is a subunit of the heteromeric transcription factor, GABP. The other subunit is GABPbeta, a notch-related protein(12) . GABP was originally identified as the transcription factor that binds to a purine-rich cis-regulatory element required for VP16-mediated activation of herpes simplex virus immediate early gene (13) . A separate set of filters was therefore analyzed using antibodies against GABPalpha or GABPbeta1. One band, identical in size to that seen using the pan-Ets antibody, is seen with anti-GABPalpha. Anti-GABPbeta1 antibody reveals two bands. The lower, more intense band migrates with an apparent molecular mass of 43 kDa and thus likely represents GABPbeta1. The levels of GABPalpha and GABPbeta1 are not significantly different in nuclear extracts from control and insulin-treated cells (data not shown).


Figure 1: Analysis of Ets-related proteins in GH4 cell nuclear extracts by immunoblotting. Lane 1, 1 µg, and lane 2, 3 µg of GH4 cell nuclear extract blotted with an antibody prepared against the conserved DNA binding domain of the c-Ets-1 (Santa Cruz). Lanes 3 and 4 are two nuclear extracts blotted against a polyclonal antibody to GABPalpha (Dr. S. L. McKnight). Lanes 5 and 6 are the same nuclear extracts blotted with a polyclonal antibody to GABPbeta1 (Dr. S. L. McKnight). The position of migration of molecular weight markers ovalbumin (46,000 kDa) and bovine serum albumin (68,000 kDa) is indicated. The arrow on the left indicates the position of GABPalpha while the arrow on the right indicates GABPbeta1.



Experiments were performed to determine if GABP might be involved in the response of the prolactin gene to insulin. Cotransfection of small amounts of GABPalpha expression vector inhibited the insulin-induced increase in prolactin gene expression (Fig. 2) to 25% of that seen in control transfections (22-fold) or in transfections with expression vector (21-fold). Cotransfection with an expression vector for GABPbeta1 resulted in a 2-fold increase in the insulin-stimulated prolactin-CAT expression (45-fold). This suggests that levels of GABPbeta1 may be limiting in GH cells. GABPbeta1 was shown to increase the affinity of GABPalpha binding to DNA(12) . Thus, it might be expected that a cotransfection with both GABPalpha and GABPbeta1 would further affect insulin-increased prolactin-CAT expression. Coexpression of vectors for both GABPalpha with GABPbeta1 completely eliminated any effect of insulin (Fig. 2) but has no significant effect on basal or EGF-increased prolactin gene expression. Basal expression of prolactin-CAT was not affected in these experiments (1.15 ± 0.25% acetylation/10 µg of protein in control cultures versus 1.07 ± 0.44% acetylation/10 µg of protein in GABPalpha + GABPbeta-cotransfected cells). Further, EGF-increased expression of prolactin-CAT was also not affected by cotransfection with GABP. EGF results in a 13-fold increase in control cells (15.12 ± 0.7% acetylation/10 µg of protein) and a 12-fold increase in GABPalpha- and GABPbeta-cotransfected cells (13.6 ± 0.66% acetylation/10 µg of protein).


Figure 2: Effect of expression of GABP on insulin-increased prolactin-CAT transcription. In control transfections, GH4 cells were cotransfected with 15 µg of Prl-(-173/+75) CAT (10) and with 5 µg of an expression vector for the human insulin receptor, pRT3HIR2 (J. Whittaker, Stony Brook) alone or with 10 µg of a cytomegalovirus expression vector pRK5. Vectors expressing GABPalpha and/or GABPbeta1 (C. C. Thompson, Carnegie Institute) or Elk-1 and SRF (R. Treisman, Imperial Cancer Research Fund, London, United Kingdom) under control of the cytomegalovirus promoter were included at 5 µg in the designated experiments. The average percent acetylation/mg of protein in control and insulin-treated cultures was determined, and the insulin incubations were compared with control levels to determine the -fold stimulation by insulin (Fold-Control). The results are from three separate experiments done in duplicate.



Similar results were seen previously with SRF and Elk-1(14) . The effects of Elk-1 and SRF were attributed to squelching caused by titration of limiting components or formation of non-functional transcription complexes(14) . The squelching of prolactin gene expression due to overexpressed GABP appears to be specific since overexpression of SRF and Elk-1 has no effect on prolactin gene expression in GH cells (Fig. 2) although the Elk-1 binding site of c-Fos is similar to the insulin-response element of the prolactin gene.

Since expression of GABP was able to block insulin responsiveness of the prolactin promoter, the binding of GABPalpha and GABPbeta1 to the insulin-response element of the prolactin gene was examined. Nuclear extracts from GH cells produced a characteristic mobility shift pattern (Fig. 3, lane 1). This gel-shift pattern was identical with that previously shown to be inhibited by low levels of non-radioactive competitor(10) . Gel-mobility shift experiments performed with bacterially expressed GABPalpha showed three retarded bands with the prolactin insulin-response element (Fig. 3, lane 2). The upper band is a complex formed with a bacterially expressed protein since it was present in extracts from unprogrammed bacteria (not shown) The other two bands are complexes containing GABPalpha. Similar complexes were previously reported to be formed between DNA and GABPalpha monomer and GABPalpha dimer(12) . Only the bacterial protein band is seen with bacterially produced GABPbeta1 (lane 3). This was expected since previous studies had shown that GABPbeta1 is not a DNA binding protein(12) . When GABPalpha and GABPbeta1 were added together (lane 4), the bands corresponding to GABPalpha were no longer visible and an abundant, more slowly migrating band was seen. This band corresponds to an abundant band seen with nuclear extracts from GH cells. The identity of this band as consisting of GABPalpha and GABPbeta1 is confirmed in the experiment shown in Fig. 3(right). Lanes 1 and 2 show the gel-mobility shift pattern with nuclear extract alone (lane 1) and nuclear extract with normal rabbit serum (lane 2). Antibodies to GABPalpha, lane 3, GABPbeta1 (lane 4), or antibodies to both GABPalpha and GABPbeta1 (lane 5) shifted this complex to one with a slower migration.


Figure 3: Binding of GABP to the prolactin promoter. Left, the proteins complexed with the P-labeled Prl-(-106/-87) were: lane 1, nuclear extract; lane 2, GABPalpha; lane 3, GABPbeta1; and lane 4, GABPalpha and GABPbeta1. The positions of migration of the GABPalpha monomer, dimer, and the GABP are indicated on the left. The migration of a nonspecific bacterial protein complex is also indicated. Right, all reactions contained 2 µg of GH4 cell nuclear extract. In addition, lane 2 contained 1 µl of normal rabbit serum, lane 3 contained a polyclonal antibody to GABPalpha (Dr. S. L. McKnight), lane 4 contained a polyclonal antibody to GABPbeta1 (Dr. S. L. McKnight), and lane 5 contained both antibodies. The position of GABP and the supershift are indicated on the left.



Since insulin receptor is a tyrosine-protein kinase that is activated by insulin binding, it is thought that activation of gene transcription by insulin may be the end product of a phosphorylation cascade. Therefore, we examined the phosphorylation of GABP in response to insulin in P-labeled GH cells. The phosphorylation of GABPalpha was increased 3-fold in 1 h in insulin-treated cells as compared with control cells (Fig. 4A). GABPbeta co-immunoprecipitated with GABPalpha in this experiment shows no increase in response to insulin. Immunoprecipitation with anti-GABPbeta1 confirms this observation. GABPbeta1 phosphorylation was not significantly increased by insulin treatment (20% above control) while the co-immunoprecipitated GABPalpha is increased 3-fold by insulin.


Figure 4: A, immunoprecipitation of GABPalpha and GABPbeta from P-labeled GH4 cells. GH cells were labeled with P and incubated with insulin for 1 h as described under ``Experimental Procedures.'' Labeled proteins were then precipitated with an antibody to GABPalpha (lanes 1 and 2) or an antibody to GABPbeta1 (lanes 3 and 4). Immunoprecipitations with control lysates are in lanes 1 and 3 while insulin-treated cell lysates are in lanes 2 and 4. The migration of GABPalpha and GABPbeta is indicated on the left while the migration of molecular weight markers is shown on the right. This experiment was repeated twice with similar results. B, insulin activation of MAP kinase. GH cells were transfected with 1 µg of a vector expressing a human influenza hemagglutinin-tagged MAP kinase and with 5 µg of pRT3HIR2. After a 24-h incubation in insulin-depleted serum containing medium, the cultures were incubated with insulin for 5 min or left untreated as controls. The cells were harvested and immunoprecipitated with anti-hemagglutinin antibody (Boehringer Mannheim) as described under ``Experimental Procedures.'' The kinase activity of the immunoprecipitated MAP kinase was assayed as described under ``Experimental Procedures'' using myelin basic protein (Sigma) as a substrate. Lane 1, MAP kinase from control cells; lane 2, MAP kinase from insulin-treated cells. C, MAP kinase phosphorylation of GABPalpha. Lanes 1-4 used immunoprecipitated MAP kinase from insulin-treated cells while lanes 5-8 contained MAP kinase immunoprecipitated from control cells. The even lanes contained GST-GABPalpha, and the odd lanes are GST protein. Lanes 3, 4, 7, and 8 show assays performed without MgCl(2) and with 2 mM EDTA. The arrow to the left indicates the position of GST-GABPalpha. The arrows on the right indicate the migration of molecular weight standards. An immunoblot of the MAP kinase in the immunoprecipitates used for this experiment is shown below each lane.



MAP kinase activation was shown to be required for several types of insulin responses in numerous systems(15) . Further, Elk-1, an Ets-related transcription factor, was shown to be activated by MAP kinase phosphorylation(16) . Pointed-P2, an Ets-related protein from Drosophila, is phosphorylated by MAP kinase in the sevenless signal transduction pathway(17) . Our studies (^2)suggest that insulin activation of prolactin gene expression in GH cells is MAP kinase-dependent since all factors that inhibit insulin-increased prolactin gene expression also inhibit MAP kinase activation. Therefore, MAP kinase activated by insulin might phosphorylate GABPalpha. The representative increase in MAP kinase activity in cell lysates from insulin-treated cells is shown (Fig. 4B). Multiple experiments show an increase of 10 ± 0.8-fold in MAP kinase activity in insulin-treated cells. GABPalpha contains three potential MAP kinase phosphorylation sites near the DNA binding domain. Therefore, GST-GABPalpha fusion protein containing the three MAP kinase phosphorylation sites was prepared and used in a kinase assay with MAP kinase immunoprecipitated from control or insulin-treated GH4 cells. GABPalpha was phosphorylated only by MAP kinase immunoprecipitated from insulin-treated cells (Fig. 4C). This shows that GABPalpha is a substrate for MAP kinase and suggests that MAP kinase phosphorylation of GABP may be functionally important.

GABP was shown to be important for enhancement of transcription of the herpes simplex virus immediate early gene, but its physiological role in uninfected cells is unknown. Our studies show that GABP mediates the insulin response of the prolactin gene. Since GABP is widely distributed, these results could be significant to understanding insulin regulation of other genes. Analysis of 22 insulin-responsive promoters has identified potential Ets-response elements in all of these. For some of these, the Ets-response element is in a region defined by deletion analysis to be important for the effects of insulin (5) . The insulin-mediated increase in the transcription of all three genes, prolactin, somatostatin, and thymidine kinase, that we have studied is inhibited by dominant-negative Ets protein. This indicates that GABP may be implicated in the regulation of other insulin-responsive genes.

Ets-related transcription factors such as GABP are often found in large complexes with other transcription factors. For example, Ets-1 and Sp-1 interact to synergistically activate the human T-cell lymphotrophic virus long terminal repeat(18) . Although this report demonstrates that GABP is necessary to the insulin effect, it may not be sufficient. The insulin responsiveness of the prolactin gene can be eliminated by mutation of two Ets motifs at -96/-87 and -76/-67 of the prolactin promoter(4) . These mutations have little effect on basal prolactin gene transcription. However, mutation of -101/-92 of the prolactin promoter eliminates the effect of insulin and reduces basal prolactin gene expression by >100-fold. Clearly, another protein(s) interacts at this sequence and is important both for basal prolactin gene expression and the effect of insulin. It is likely that GABP is complexed with this protein(s) in the prolactin promoter and that this complex is important to the increase in prolactin gene expression seen in insulin-treated cells.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK43365. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Mt. Sinai Medical Center, Asher Levy Place, New York, NY 10029.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; EGF, epidermal growth factor; SRF, serum response factor.

(^2)
J. Sap, K. K. Jacob, and F. M. Stanley, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank S. L. McKnight, C. C. Thompson, R. Treisman, and J. Whittaker for plasmids and antibodies used in these studies.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.