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Keywords:

  • Proenkephalin;
  • Src kinase;
  • Cyclic AMP response element-binding protein;
  • Forskolin;
  • Gene regulation

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

Abstract: The transcription factor CREB [cyclic AMP response element (CRE)-binding protein] is activated by several kinase pathways on phosphorylation of serine-133. Phosphorylation of CREB at serine-133 is required for the induction of target gene expression. The proenkephalin gene is a target of cyclic AMP-dependent agonists like forskolin, and its expression is driven by the enhancer element CRE-2. It has been shown that CREB binds CRE-2 in extracts from striatum and hypothalamus. However, these studies did not show a functional requirement for CREB serine-133 phosphorylation in CRE-2 function. We demonstrate that CREB binds CRE-2 in primary astrocyte cultures and that transcriptional activation of CRE-2 requires CREB phosphorylation at serine-133. In addition, it has recently been shown that, at least in some contexts, CREB phosphorylation is not sufficient to activate target gene expression and that another intracellular signal seems to be required. Therefore, we also sought to determine if another signaling event, in addition to CREB phosphorylation, might be involved in cyclic AMP-mediated induction of the proenkephalin gene. We have found that the inhibition of src-related nonreceptor tyrosine kinases blocks forskolin-induced proenkephalin gene expression without having any effect on serine-133-phosphorylated CREB levels and that constitutively activated src kinase can activate the proenkephalin promoter.

The proenkephalin gene is up-regulated in various cell types in culture and in vivo on activation of several intracellular signaling pathways, including the cyclic AMP (cAMP)-dependent signaling pathway. The cAMP-activated signaling pathway induces this gene via an enhancer that is composed of multiple protein binding elements (Comb et al., 1986; Hyman et al., 1989; Nguyen et al., 1990). Mutational analysis of the proenkephalin enhancer has defined a 7-bp sequence (TGCGTCA), referred to as cAMP response element-2 (CRE-2), that is required for functional activity (Comb et al., 1988). CRE-2 differs by two bases from the prototypical binding site for AP-1 proteins (TGACTCA) and by one base from a symmetric binding site (TGACGTCA) for the CRE-binding protein (CREB). Studies using in vitro translated transcription factors have demonstrated that CREB/ATF, Jun/Fos, and some C/EBP complexes can bind to this element (Sonnenberg et al., 1989; Kobierski et al., 1991; Chu et al., 1994; Vallejo et al., 1993). However, in vivo studies have shown that CREB is the CRE-2-binding protein in extracts from striatum and hypothalamus (Konradi et al., 1993, 1994; Borsook et al., 1994).

CREB is transcriptionally activated when phosphorylated at serine-133 (P-CREB) by several serine/threonine kinases, including protein kinase A (PKA). Phosphorylation of CREB at serine-133 increases its association with CREB-binding protein (CBP) and results in increased target gene expression. Manipulations that block CREB phosphorylation at serine-133 or CREB:CBP interactions also block CREB-mediated target gene expression (Gonzalez and Montminy, 1989; Sheng et al., 1991; Arias et al., 1994; Kwok et al., 1994). However, some recent studies suggest that although P-CREB is required for CBP binding and/or subsequent changes in target gene expression, it may not be sufficient. For example, T cell receptor stimulation of Jurkat cells induces high levels of P-CREB but requires costimulation with suboptimal concentrations of a cAMP agonist to induce CREB:CBP interactions and target gene expression (Brindle et al., 1995). Induction of CRE activity by voltage-sensitive Ca2+ channels is markedly reduced in a PKA-deficient PC12 cell line despite the fact that CREB phosphorylation at serine-133 occurs as normal (Thompson et al., 1995).

Such studies suggest that stimulation of PKA generates an as yet unidentified signal that is required in addition to P-CREB for full induction of CRE-driven target gene expression. It is well established that cAMP can regulate other kinases in addition to PKA. For example, elevated levels of cAMP lead to a rapid stimulation of the atypical protein kinase C isoform ζ (Wooten et al., 1996). cAMP inhibits mitogen-activated protein kinases in some cell types but activates them in others (Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Faure et al., 1994; Frodin et al., 1994; Calleja et al., 1997). In addition, the nonreceptor tyrosine kinase src also can be induced by cAMP analogues (Roth et al., 1983). Src kinase activity is negatively regulated when phosphorylated at tyrosine-527 by C-terminal src kinase (Brown and Cooper, 1996). An indirect mechanism of src activation by PKA is suggested by the finding that PKA can phosphorylate and inactivate C-terminal src kinase (Brown and Cooper, 1996; Sun et al., 1997).

In this study we have sought to determine if another kinase pathway, in addition to PKA and the resultant induction of P-CREB, is playing a role in cAMP-mediated target gene expression. Using proenkephalin as a target gene we establish that CREB binds to the proenkephalin enhancer in primary cortical astrocyte cultures and that phosphorylation of CREB at serine-133 is required for forskolin-mediated induction of the proenkephalin promoter. A src kinase inhibitor blocks forskolin-mediated increases of the proenkephalin gene as well as both proenkephalin and somatostatin levels without any apparent effects on P-CREB levels. However, src kinases do not appear to act independently of P-CREB because mutation of CREB serine-133 blocks src-induced activation of the proenkephalin promoter. Although further analysis is required to determine the exact role of src kinases in proenkephalin gene induction, several possibilities are discussed.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

Primary cell culture

Cortex was dissected from Sprague-Dawley neonatal rats into Hanks’ balanced salt solution. Cortical tissue was mechanically dissociated with a fire-narrowed Pasteur pipette, and dissociated cells were plated in cell culture medium [90% Dulbecco’s modified Eagle’s medium/F12 (without sodium bicarbonate), 10% fetal bovine serum, 0.15% sodium bicarbonate, 5 units/ml penicillin, and 5 mg/ml streptomycin] at a density of 1 × 107-4 × 107 cells per 175-cm2 tissue culture flask. At 7-10 days after plating confluent cultures were shaken at 200 rpm overnight (15-20 h) and subsequently were covered with fresh medium after two washes with phosphate-buffered saline (PBS). According to Melner et al. (1990), this procedure removes oligodendrocyte type 2-astrocyte lineage cells and microglia. In some cases cultures were then shaken for an additional overnight period or were incubated for 3 h in medium containing 3 mM carbonyl iron. Both of these procedures are intended to remove any remaining microglia from the cultures (Melner et al., 1990; Giulian et al., 1993).

Cortical astrocyte cultures were plated at a density of 4 × 105 cells per well in six-well plates or 1 × 106 cells per 10-cm plate and grown to confluence with medium changes every 3 days. Low-serum (0.1%) medium was added 1-2 days before treatment with 10 μM forskolin (Sigma). Some cultures were pretreated for 1 h with the following kinase inhibitors: H-89 (7 μM; Calbiochem), herbimycin A (8.7 μM; Calbiochem and GibcoBRL), or bisindolylmaleimide (100 nM; Calbiochem).

Nuclear extracts and electrophoretic mobility shift assays

Cells were harvested in 500 μl of cold PBS, pelleted, resuspended in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.6% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate], and incubated (at 0°C) for 15 min. Crude nuclear extracts were made from resultant pellets after resuspension in buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 0.4 μM microcysteine, 12.5% glycerol, and leupeptin and aprotinin (5 μg/ml each)] and incubation at 0°C for 15 min.

For electrophoretic mobility shift assays a double-stranded oligonucleotide containing the CRE-2 consensus site (5′-GATCGGCCTGCGTCAGCTG-3′) was labeled using Superscript reverse transcriptase (GibcoBRL) and [α-32P]dCTP (3,000 Ci/mmol). Four microliters of nuclear lysate and 1 ng of radiolabeled probe were then mixed with 15 μl of binding buffer containing 8 mM HEPES (pH 7.9), 20 mM potassium phosphate (pH 7.9), 8% glycerol, 0.3 mM EDTA, 1.2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 μg of dIdC at 0°C for 20 min. For supershift experiments a CREB-specific antibody (Upstate Biotechnology) was mixed with lysate and binding buffer (1 μl of antibody solution per reaction) for 30 min at 0°C before addition of radiolabeled probe.

Northern blot analysis

Cortical astrocyte cultures were plated at a density of 1 × 106 cells per well in six-well plates and grown to confluence with medium changes every 3 days. Low-serum (0.1%) medium was added 1-2 days before treatment with 10 μM forskolin (Sigma). Some cultures were pretreated for 1 h with the following kinase inhibitors: H-89 (7 μM; Calbiochem), herbimycin A (8.7 μM; Calbiochem and GibcoBRL), or bisindolylmaleimide (100 nM; Calbiochem). After forskolin treatments of 5-6 h, total RNA was isolated using the Nonidet P-40 lysis method, separated by size on a 1.4% denaturing agarose gel, and electroblotted onto a nylon membrane as described by Cole et al. (1995). Membrane-bound RNA was then hybridized with a rat proenkephalin riboprobe or a cyclophilin cDNA probe (Cole et al., 1995) and visualized with a PhosphorImager (Molecular Dynamics).

Preparation of protein samples and western blot analysis

Cultures were treated with forskolin over an interval of 15 min-2 h, and whole cell extracts were collected in a sodium dodecyl sulfate (SDS)/gel loading buffer (pH 6.7) containing 2% SDS, 5% β-mercaptoethanol, 4% glycerol, and 50 mM Tris. Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using a 10% acrylamide concentration, and proteins were then transferred to Immobilon-P membranes (Millipore). Membranes were blocked for 1 h in a solution of 5% milk and 0.05% Tween 20 in PBS. Unless stated otherwise, the milk concentration was reduced to 0.5% for all subsequent antibody incubations and washes. Membranes were incubated with primary antibody, anti-P-CREB antibody (1:1,000; Upstate Biotechnology), for 1 h at room temperature. After three 10-min washes, membranes were then incubated with a horseradish peroxidase-linked donkey anti-rabbit antibody (1:1,000; Amersham Life Science) for 1 h at room temperature. Membranes then were washed once in PBS containing 0.05% Tween 20 and twice in PBS alone. Horseradish peroxidase-containing antibody complexes were then visualized using Renaissance chemiluminescence reagents (NEN).

Transient transfection and chloramphenicol acetyltransferase (CAT) assays

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

For transient transfections cortical astrocyte cultures were plated at a density of 3 × 105 cells per well in six-well plates. The next day medium was replaced with half as much fresh medium, and DNA precipitates were prepared by standard calcium phosphate methods. All plasmids have been previously described: pENKAT-12 (Comb et al., 1986); 71SRIFCAT (Yamamoto et al., 1988); v-src (Johnson et al., 1985); and pG5ENKΔ80, pGAL4-CREB, and pGAL4-CREB(ala-133) (Tan et al., 1996). All precipitates contained 2 μg of the cytomegalovirus (CMV)-β-galactosidase construct pCMVβ-galactosidase from GibcoBRL as an internal control and 2 μg of pENKAT-12, p-71SRIFCAT, or pG5ENKΔ80. In some cases cotransfections also included a plasmid that constitutively expresses v-src. pG5ENKΔ80 also was cotransfected with 2 μg of pGAL4-CREB or pGAL4-CREB(ala-133). Total DNA concentration was maintained at 8 μg with pGEM. At 4 h after addition of precipitates, cells were washed once with PBS and covered with low-serum medium. At 15-18 h later the cells were treated with 10 μM forskolin for 5-6 h. When used, herbimycin A was added 1 h before forskolin. Cytoplasmic lysates were collected in the following buffer: 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 0.1% Triton X-100, and 1 mM dithiothreitol. CAT assays and β-galactosidase assays were done as described using [14C]chloramphenicol (Nguyen et al., 1990). The mean and SD for each data point were determined from three transfections using Statworks 1.2 (Macintosh).

CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

Previous studies have demonstrated that CREB is the CRE-2-binding protein in nuclear extracts obtained from rat brain striatum and hypothalamus (Konradi et al., 1993, 1994, 1995; Borsook et al., 1994). However, studies using in vitro translated proteins raise the possibility that other transcription factors may play a role under other conditions or in different cell types (Sonnenberg et al., 1989; Kobierski et al., 1991; Chu et al., 1994; Vallejo et al., 1993). To clarify the role of CREB at the proenkephalin promoter in cultured rat cortical astrocytes, nuclear extracts were made and then incubated with a double-stranded oligonucleotide probe containing the CRE-2 sequence. Using electrophoretic mobility shift assays we show that protein complexes with two different mobilities bind to CRE-2 constitutively and that neither the intensity nor the mobility of the CRE-2-binding proteins changes with forskolin treatment (Fig. 1A). To determine if either of these complexes contains CREB protein, extracts were incubated with labeled CRE-2 and a CREB-specific antibody (from Upstate Biotechnology). As shown in Fig. 1A, right panel, anti-CREB antibody mostly blocked and partly supershifted the more slowly migrating upper band.

image

Figure 1. CREB activates the proenkephalin enhancer in cortical astrocyte cultures. A: Cortical astrocyte nuclear extracts were incubated with a radiolabeled double-stranded oligonucleotide probe containing the proenkephalin CRE-2 sequence and analyzed in gel mobility shift assays. To determine if CREB binds to CRE-2, CREB specific antibody was added (right panel). Binding of the more prominent complex (indicated by arrow) was blocked. forsk., forskolin. B: Astrocyte cultures were transiently transfected with 2 μg of an enkephalin-CAT reporter plasmid, pENK12, and with 3 μg of the following expression vectors—pRSVKCREB, CREB with a mutation in the DNA binding domain (KCREB), or pRSVCREBser-133, CREB with a mutation at serine-133 (P-CREBm), as indicated. Following transfection cultures were exposed to 10 μM forskolin for 18 h as indicated. Changes in CAT activity are expressed as a percentage of forskolin induction. Data are mean ± SD (bars) values of three experiments. SD values were calculated using Statworks.

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To confirm that CREB is required for transcriptional activation of the proenkephalin promoter, we cotransfected the proenkephalin enhancer-CAT fusion plasmid (pENK12) with plasmids that constitutively express one of two functionally inactive mutant CREB proteins: P-CREBm, in which serine-133 is converted to an alanine, or KCREB, which cannot bind to DNA owing to a point mutation in its DNA binding domain (arginine-287 to leucine) (Walton et al., 1992). In each case the reporter construct was cotransfected with a CMV-β-galactosidase construct as a control to which the data are normalized. Consistent with previous studies, activity of the proenkephalin enhancer/promoter was induced from four- to 10-fold by 10 μM forskolin (Comb et al., 1986; Hyman et al., 1989; Kobierski et al., 1991). For statistical analysis of the effects of cotransfected plasmids, forskolin induction was set at 100%, and the effects of cotransfected plasmids are expressed as percent reductions of this forskolin induction. Mean and SD values are shown and calculated from three data points. As shown in Fig. 1B cotransfection of P-CREBm reduced the forskolin induction of pENK12 to 25%. KCREB was even more effective at blocking the forskolin induction of pENK12, leading to a reduction in pENK12 activation that is only 9% of the induction seen with forskolin alone (Fig. 1B). Collectively, these data show that CREB binds to CRE-2 and plays a role in transcriptional activation of the enhancer in forskolin-stimulated astrocytes.

A src-specific tyrosine kinase inhibitor blocks forskolin-induced proenkephalin gene expression

Previous studies have demonstrated cross talk between PKA and other kinase pathways (Roth et al., 1983; Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Faure et al., 1994; Frodin et al., 1994; Wooten et al., 1996; Calleja et al., 1997; Sun et al., 1997). To determine if another kinase pathway, in addition to PKA, might play a role in cAMP-dependent increases in proenkephalin gene expression, cortical astrocyte cultures were treated with several kinase inhibitors of varying specificity before addition of forskolin and were subsequently harvested to analyze levels of proenkephalin mRNA. As shown in Fig. 2A, treating astrocytes with 10 μM forskolin results in a strong increase in proenkephalin mRNA levels. As expected, H-89, an inhibitor of PKA, completely blocked the forskolin-dependent induction of proenkephalin mRNA measured by northern blot (Fig. 2A). Cells also were pretreated with an inhibitor of the src family of tyrosine kinases, herbimycin A. Like H-89, herbimycin A was able to block fully the forskolin-induced induction of proenkephalin mRNA (Fig. 2A). In addition, cells were pretreated with a protein kinase C inhibitor (bisindolylmaleimide) and a Ca2+/calmodulin-dependent kinase II inhibitor (KN-62). Neither of these compounds had any effect on forskolin-induced changes in proenkephalin gene expression (Fig. 2A). In addition, herbimycin A alone had no effect on basal levels of proenkephalin expression (data not shown). To confirm that the effect of herbimycin A was specific, cyclophilin RNA levels were measured, and levels of proenkephalin RNA were normalized to those of cyclophilin using a Molecular Dynamics PhosphorImager. Mean and SD values are shown and were calculated from three experiments. As shown in Fig. 2B proenkephalin levels are elevated three- to 10-fold by forskolin, with a median fold induction of approximately sevenfold as determined using the Macintosh program Statview. Also shown in Fig. 2B is the finding that the forskolin induction of proenkephalin is blocked by herbimycin after normalization to cyclophilin.

image

Figure 2. Effects of kinase inhibitors on forskolin (forsk.)-induced proenkephalin gene expression. A: Astrocyte cultures were treated with the following kinase inhibitors as indicated—7 μM H-89, 8.7 μM herbimycin A (Herb. A), 100 nM bisindolylmaleimide (BIM), or 1.5 μM KN-62. One hour later cells were treated with 10 μM forsk. for an additional 5 h. Total cytoplasmic RNA was isolated and analyzed by northern blot analysis with a riboprobe specific for proenkephalin mRNA. B: RNA membranes were reprobed for cyclophilin, and levels of proenkephalin (ENK) RNA were normalized to those of cyclophilin using a Molecular Dynamics Phosphorlmager. Data are mean ± SD (bars) values from three experiments.

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In addition, we also transfected cultured primary astrocytes with two CRE-driven reporter gene constructs: pENK12 and p-71SRIF (somatostatin enhancer-CAT fusion reporter). Herbimycin A was added to the cultures 1 h before forskolin, and cells were exposed to forskolin (in the presence of herbimycin A) for 5 h. In each case the reporter construct was cotransfected with a CMV-β-galactosidase construct as a control to which the data are normalized. As expected, activity of both pENK12 and p-71SRIF was induced from four- to 10-fold by 10 μM forskolin. For statistical analysis of the effect of herbimycin A, forskolin induction was set at 100%, and the effect of herbimycin A is expressed as a percent reduction of the forskolin effect. Mean and SD values are shown and calculated from three sets of data. As shown in Fig. 3 pretreatment with herbimycin A reduced the forskolin-mediated activation of pENK12 and p-71SRIF to slightly <25% of the forskolin-alone value.

image

Figure 3. Herbimycin A (Herb. A) blocks the forskolin induction of two CRE-driven promoters. A: Astrocyte cultures were transiently transfected with 2 μg of an enkephalin-CAT reporter plasmid (pENK12) and 2 μg of CMV-β-galactosidase. Following transfection some cultures were exposed to 10 μM forskolin for 5 h as indicated. Herb. A (8.7 μM) was added 1 h before forskolin as indicated. B: Astrocyte cultures were transiently transfected with 2 μg of a somatostatin-CAT reporter plasmid (p-71SRIF) and 2 μg of CMV-β-galactosidase. Following transfection some cultures were exposed to 10 μM forskolin for 5 h as indicated. Herb. A (8.7 μM) was added 1 h before forskolin as indicated. In A and B, changes in CAT activity are expressed as a percentage of forskolin induction and are normalized to β-galactosidase activity. Data are mean ± SD (bars) values of three data points. SD values were calculated using Statworks.

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Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

Given that forskolin-mediated activation of proenkephalin requires P-CREB and that herbimycin A inhibits forskolin-mediated induction of proenkephalin RNA and the proenkephalin and somatostatin reporter plasmids, we asked whether herbimycin A can block forskolin-mediated P-CREB induction. To address this point, whole-cell extracts were made from cells treated with forskolin for 15 min and in some cases pretreated with kinase inhibitors. The extracts were then probed by western blot analysis with an antibody that specifically recognizes P-CREB. As shown in Fig. 4 (upper panel) unstimulated astrocyte cultures have detectable levels of P-CREB that are markedly increased within 15 min of forskolin treatment. As expected, H-89 blocked the forskolin-mediated elevation of P-CREB content measured in whole-cell extracts, whereas KN-62 and bisindolylmaleimide had no effect (Fig. 4). However, herbimycin A at a concentration that is sufficient to block forskolin-mediated induction of the proenkephalin gene had no effect on the induction of P-CREB by forskolin (Fig. 4). To verify that changes in P-CREB levels (or lack thereof) were due to changes in CREB phosphorylation and not due to differences in amounts of CREB protein, membranes were stripped of P-CREB antibody and reprobed with an anti-CREB antibody (Fig. 4, lower panel).

image

Figure 4. Effects of kinase inhibitors on forskolin (forsk.)-stimulated phosphorylation of CREB at serine-133. Astrocyte cultures were treated with the following kinase inhibitors as indicated: 7 μM H-89, 8.7 μM herbimycin A (Herb. A), 100 nM bisindolylmaleimide (BIM), or 1.5 μM KN-62. One hour later cells were treated with 10 μM forsk. for an additional 15 min. Upper panel: Proteins in whole-cell extracts were separated by 10% SDS-PAGE and probed by western blot analysis with an anti-P-CREB-specific antibody. Lower panel: The above membrane was reprobed with an anti-CREB-specific antibody. Protein-antibody complexes were visualized using Renaissance chemiluminescence reagents (NEN).

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Several studies have shown that P-CREB levels remain elevated on a time scale of hours in cell culture and can be prolonged by certain stimuli in vivo (Brindle et al., 1995; Cole et al., 1995; Bito et al., 1996; Tan et al., 1996). Treatments that prolong P-CREB induction also up-regulate target gene expression, suggesting that the duration of CREB phosphorylation at serine-133 provides an important signal for downstream gene regulation (Bito et al., 1996). To determine if herbimycin A blocks forskolin-induced proenkephalin gene activation by shortening the time course of P-CREB stimulation, cells were pretreated with H-89 or herbimycin A, and P-CREB levels were measured over an extended time course (up to 4 h). In the absence of kinase inhibitors, maximal levels of P-CREB were reached within 15 min of treatment with forskolin; P-CREB levels remained elevated up to 2 h later and dropped to baseline levels by 4 h (Fig. 5A and B). As shown in Fig. 5A, the PKA inhibitor H-89 blocked P-CREB induction by forskolin at all timepoints shown (15 min up to 2 h). In contrast, a concentration of herbimycin A that is sufficient to block induction of proenkephalin mRNA and pENK12 and p-71SRIF plasmids by forskolin (see Figs. 2 and 3) did not block forskolin-mediated P-CREB induction at any timepoint shown (compare Fig. 5B and C).

image

Figure 5. Herbimycin A has no effect on forskolin (forsk.)-stimulated phosphorylation of CREB at serine-133. Astrocyte cultures were treated with (A) 7 μM H-89, (B and C) 8.7 μM herbimycin A (Herb. A). One hour later cells were treated with 10 μM forsk. for intervals ranging from 15 min up to 4 h. Proteins in whole-cell extracts were separated by 10% SDS-PAGE and probed by western blot analysis with an anti-P-CREB-specific antibody.

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CRE/CREB-driven promoters can be activated by v-src

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

We have shown that CREB binds the proenkephalin CRE-2 element in cortical astrocytes and that a mutation at CREB serine-133 blocks induction of the proenkephalin promoter by forskolin. However, the src kinase inhibitor herbimycin A can block forskolin-mediated increases in proenkephalin gene expression and induction of two CRE-driven promoters in transient transfection assays with no apparent effect on P-CREB levels. To establish further a role for src-related kinases in CRE-mediated gene expression, we cotransfected three different CRE/CREB-driven reporter plasmids with a plasmid that constitutively expresses v-src [pEVXv-src (Johnson et al., 1985)]. Two of these reporters, pENK12 and p-71SRIF, were used in Fig. 3. Transfections included a CMV-β-galactosidase construct as a control to which the data are normalized. Mean and SD values are shown and calculated from three data points. As shown in Fig. 6A and B, CAT expression is induced ∼4.5- and threefold at pENK12 and p-71SRIF, respectively, after cotransfection with v-src plasmid. Transfection of just the vector into which v-src is cloned (pEVX) had no effect on the reporter plasmid expression (data not shown).

image

Figure 6. CRE/CREB-driven promoters can be activated by v-src. A and B: Astrocyte cultures were transiently transfected with 2 μg of pENK12 (A) or p-71SRIF (B) and with 4 μg of a plasmid that expresses a constitutively active v-src protein (v-src). C: Astrocyte cultures were transiently transfected with 1.5 μg of pENKΔ80 and the same concentration of pGAL-CREB or pGAL-CREB(ala-133). As indicated, cells were transfected with 4 μg of a plasmid that expresses a constitutively active v-src protein (v-src). In A-C, transfections also included 2 μg of CMV-β-galactosidase as internal control. Changes in CAT activity are expressed as fold induction over control, which is normalized to 1. Data are mean ± SD (bars) values of three data points and were normalized to β-galactosidase activity. SD values were calculated using Statworks.

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The third reporter plasmid, pG5ENKΔ80, was used to eliminate further complications due to other endogenous factors that might interact at the proenkephalin CRE. This plasmid is a proenkephalin promoter/CAT reporter plasmid in which the proenkephalin enhancer has been removed and replaced with four copies of a GAL4 binding site (Tan et al., 1996). We coexpressed this reporter plasmid with a GAL4-CREB fusion protein that contains serine-133, pGAL4-CREB. Tan et al. (1996) have shown that cAMP- and fibroblast growth factor-mediated activation of pG5ENKΔ80 requires a functional serine-133 in pGAL4-CREB. We show in Fig. 6C that in the presence of GAL4-CREB, v-src activates pG5ENKΔ80 expression approximately threefold. In addition, if pGAL4-CREB is replaced by a construct in which CREB serine-133 is mutated to an alanine, pGAL4-CREB(ala-133), there is no significant activation of pG5ENKΔ80 by v-src (Fig. 6C). These data show that CREB phosphorylation is essential for src function in this context.

DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

Proenkephalin is a long-established target of cAMP-dependent agonists like forskolin, and its expression is driven by the enhancer element CRE-2. The first goal of this study was to determine if CREB is the CRE-2-binding protein in our primary cortical astrocyte cultures and to confirm that the transcriptional activation of CRE-2 requires CREB phosphorylation at serine-133. Consistent with previous studies, nuclear extracts from cortical astrocytes contain two complexes that bind to CRE-2 (Konradi et al., 1993, 1994; Borsook et al., 1994). The binding of the upper, more slowly migrating complex is blocked or supershifted by a well-characterized CREB-specific antibody, confirming that this complex consists of CREB protein. Our transfections with a CREB serine-133 mutant and a CREB DNA-binding mutant also establish that phosphorylation at serine-133 (P-CREB) is required for CRE-2 activation.

We also were interested in identifying intracellular signaling molecules/pathways that are required, in addition to P-CREB, for forskolin-mediated target gene induction. Because previous studies have shown that cAMP levels can modulate other kinase pathways in addition to PKA, we asked whether non-PKA kinases might play a role in the regulation of a CRE-driven target gene. Predictably, a PKA inhibitor, H-89, completely blocked forskolin-mediated increases in proenkephalin mRNA levels. In addition, herbimycin A, an inhibitor specific for the src family of tyrosine kinases, also blocks the induction of proenkephalin by forskolin and the forskolin stimulation of a proenkephalin reporter gene, pENK12, and the somatostatin reporter construct, p-71SRIF. The effects of herbimycin A on p-71SRIF are especially interesting because the somatostatin CRE is a palindromic CREB homodimer binding site.

An alternative interpretation of the herbimycin A effect could be that herbimycin A is nonspecifically inhibiting PKA. However, it has previously been shown that concentrations of herbimycin A up to 40 times higher than that used here do not block PKA-dependent phosphorylation in vitro (Fukazawa et al., 1991). Furthermore, we also show that herbimycin A does not block forskolin-induced phosphorylation of CREB at serine-133, which is known to be mediated by PKA activation.

A role for src kinases in CRE-mediated gene expression is further supported by our observations that cotransfection of a v-src plasmid can induce transcriptional activity from three CRE/CREB reporter plasmids. One of these reporter plasmids, pG5ENKΔ80, cannot be activated by endogenous transcription factors because its CRE is replaced by four GAL4 binding sites. Its activation is dependent on coexpression of a GAL4 fusion protein that contains the serine-133 domain of CREB. Therefore, activation of this construct by v-src further supports the argument that src is acting via a mechanism that involves P-CREB. Experiments with the src kinase inhibitor herbimycin A suggest that src does not itself increase P-CREB levels, so we presume that v-src acts in conjunction with basal levels of P-CREB and basal levels of phosphorylated GAL4-CREB protein.

The src family of tyrosine kinases has nine members, and many are expressed ubiquitously. These kinases are nonreceptor, membrane-bound tyrosine kinases. Their activity is induced by dephosphorylation of tyrosine-527 and further enhanced by phosphorylation of tyrosine-416 (Brown and Cooper, 1996; Sicheri et al., 1997; Xu et al., 1997). Src kinases are activated by several extracellular agents, including growth factors, oxidative- and UV-induced stress, and certain G protein-coupled receptors (Ralston and Bishop, 1985; Devary et al., 1992; Simonson and Herman, 1993; Chen et al., 1994; Mukhopadhyay et al., 1995; Diverse-Pierluissi et al., 1997). Once activated, they mediate a wide range of cellular functions, including gene regulation (Xie and Herschman, 1995; Brown and Cooper, 1996; Szabo et al., 1996; Campbell et al., 1997; Yu et al., 1997). Another study has shown that cAMP analogues can induce src kinase activity, and an indirect mechanism of activation is suggested by the observation that cAMP inhibits the src-inhibiting kinase C-terminal src kinase (Roth et al., 1983; Sun et al., 1997). In addition, src kinase activity can be stimulated by an inhibitor of serine/threonine phosphatases (Chackalaparampil et al., 1994). Src also contains a PKA consensus site at serine-17; however, to date, this site has not been associated with kinase function (Yaciuk et al., 1989).

Our results support the hypothesis that src-related kinases play a role in cAMP-mediated induction of a CRE-driven target gene but do not identify the mechanism of action. However, some pathways of action are less likely than others. For example, it is unlikely that CREB is a direct target of src because CREB phosphorylation has been carefully studied, and no increases in tyrosine phosphorylation have been reported (Yamamoto et al., 1988; Lee et al., 1990; Sheng et al., 1990). In our own studies of forskolin-treated astrocytes, we have detected no increases in CREB tyrosine phosphorylation either (data not shown). Alternatively, src kinases might indirectly increase proenkephalin gene expression via effects on pathways that control cAMP accumulation or dephosphorylation of P-CREB. If this were the case in our astrocyte cultures, one might predict that src kinase inhibition would block/decrease induction of P-CREB or shorten the duration of the signal. However, pretreatment with the src kinase inhibitor herbimycin A had no effect on P-CREB induction by forskolin. Overexpression of c-src does phosphorylate G α subunits and thereby enhances the β-adrenergic-stimulated accumulation of cAMP (Bushman et al., 1990; Hausdorff et al., 1992; Moyers et al., 1993). However, in this study we used forskolin, which is a direct activator of adenylyl cyclase that acts downstream of receptor-coupled G proteins. Sun and Maurer (1995) have identified a second phosphorylation site on CREB (serine-142) that negatively regulates CREB activity when phosphorylated by calcium/calmodulin-dependent kinase II. Our observation that KN-62, a specific inhibitor of calcium/calmodulin-dependent kinase II, had no effect on forskolin-induced proenkephalin gene expression suggests that src kinases are not regulating CRE-driven gene expression indirectly via phosphorylation at this residue. Given that src kinases do not seem to be required for forskolin-mediated induction of CREB phosphorylation, it could be hypothesized that src kinases are part of a PKA-dependent pathway that acts in parallel to rather than with P-CREB. However, the induction of pG5ENKΔ80 by v-src argues that this is not the case.

Alternatively, we suggest that src kinases generate a second signal that works in conjunction with CREB phosphorylation to mediate cAMP-induced target gene expression. This interpretation is consistent with several recent reports in the literature (Brindle et al., 1995; Thompson et al., 1995). It was demonstrated in one of these studies that stimulation of Jurkat TCR-CD3 T cell receptor complexes induces P-CREB but does not promote association of CREB with CBP and does not induce target gene expression. However, if TCR-CD3 activation is combined with subthreshold concentrations of a cAMP agonist, the formation of CREB:CBP complexes is induced, and target gene expression increases in the absence of any additional effects on P-CREB (Brindle et al., 1995). The cAMP agonist in this case seems to stimulate a second signal that acts together with P-CREB to induce CREB:CBP interactions. In addition to their interactions with each other, CBP and CREB also bind to components of the general transcription complex. CREB interacts with a component of TFIID (dTAFII110), and CBP binds to RNA polymerase II and TFIIB; each of these interactions has been shown to be important for transcriptional activation by CREB (Ferrari et al., 1994; Kowk et al., 1994; Kee et al., 1996).

We conclude that src kinases play a role in cAMP-dependent target gene induction that is separate from but also dependent on phosphorylation of CREB at serine-133. It is not clear at this point the exact level of src function in CRE-driven target gene expression. It will be of great interest to determine if src is required for key protein-protein interactions between CREB and CBP or between CREB/CBP and general transcription factors or an as yet identified component of this signaling pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References

We thank Dr. Richard Goodman for CREB expression plasmids, Dr. Joan Brugge for src expression plasmids, and Dr. Yi Tan for GAL4 vectors. We also thank Susan Lewis for a critical reading of the manuscript. This work was supported by grant DA 10160 from the National Institute on Drug Abuse (to S.E.H. and D.B.) and pilot grant AG05134 from the Massachusetts Alzheimer’s Disease Research Center (to L.A.K.).

References

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Transient transfection and chloramphenicol acetyltransferase (CAT) assays
  5. RESULTS
  6. CREB/P-CREB activates the proenkephalin enhancer in cortical astrocyte cultures
  7. Herbimycin A does not inhibit forskolin-stimulated phosphorylation of CREB at serine-133
  8. CRE/CREB-driven promoters can be activated by v-src
  9. DISCUSSION
  10. Acknowledgements
  11. References
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