Serine 236 of hERα is phosphorylated by PKA.
Previous studies have indicated that phosphorylation of hERα is increased by the activation of PKA (see references 21
). In order to directly determine whether PKA can phosphorylate hERα in vitro, we transfected COS-1 cells with an expression vector (pSG5) containing the open reading frame encoding hERα (HEG0). At 48 h after transfection the cells were harvested, WCEs were prepared, and 100 μg of the WCEs were immunoprecipitated with monoclonal antibody F3. Immunoprecipitates were incubated with the purified catalytic subunit of PKA in the presence of [γ-32
P]ATP, followed by resolution by SDS-PAGE and autoradiography. Incubation of the immunoprecipitates with PKA resulted in phosphorylation of HEG0 (Fig. A, lane 2).
FIG. 2 Serine 236 is phosphorylated by PKA. (A) WCEs of COS-1 cells transiently transfected with HEG0, HEG19, or HE15 were immunoprecipitated with F3 (lanes 1 and 2) and B10 (lane 3) monoclonal antibodies, phosphorylated with PKA, and analyzed by SDS-PAGE and (more ...)
hERα deletion mutants lacking either the N-terminal A/B region (HEG19) or the LBD and region F (HE15) (Fig. A) were transfected into COS-1 cells, and WCEs were immunoprecipitated with monoclonal antibodies F3 or B10, respectively, and phosphorylated with the catalytic subunit of PKA. Both HEG19 and HE15 were phosphorylated by PKA in vitro (Fig. A, lanes 1 and 3), indicating either that PKA phosphorylates hERα within or near the DBD and/or that hERα is phosphorylated by PKA at sites in the A/B region and in the LBD.
FIG. 1 Schematic representation of hERα. (A) Regions A to F, as initially described by Krust et al. (49) are depicted, the numbers below referring to amino acid positions of the boundaries for the different regions. Also shown are the positions of the (more ...)
Examination of the amino acid sequence of hERα showed that two serine residues, serine 236 (NRRKS
C) in the DBD (Fig. B) and serine 305 (SKKNS
L) in the LBD could be potential PKA phosphorylation sites (53
). Only one of these sites (serine 236) is present in both HE15 and HEG19 and was mutated to the nonphosphorylatable residue alanine. Immunoprecipitates were incubated with PKA as described above, resolved by SDS-PAGE, and transferred to nitrocellulose. Immunoblotting with B10 was performed to compare levels of HE15 and HE15236A
protein, and autoradiography of the nitrocellulose membrane showed levels of phosphorylation. HE15236A
was phosphorylated to a lower level than HE15, indicating that serine 236 is a substrate for PKA in vitro (Fig. B). HE15 was also phosphorylatable by PKC in vitro, albeit to a much lower degree. HE15 and HE15236A
were phosphorylated to a similar extent by PKC (Fig. B, lanes 5 and 6, and data not shown). No signal was obtained when PKA or PKC were omitted from the kinase assay (data not shown).
Two-dimensional phosphopeptide mapping of in vitro-phosphorylated immunoprecipitates showed the absence of three spots (dotted) from HE15236A (Fig. D) compared with HE15 (Fig. C), indicating that serine 236 is phosphorylated by PKA in vitro. The two strong spots in both the HE15 and HE15236A phosphopeptide maps suggest the presence of at least one other major PKA site resides within amino acids 1 to 281 of hERα (HE15).
We next examined the ability of PKA to alter the phosphorylated state of wild-type hERα (HEG0) in vivo. COS-1 cells transiently transfected with HEG0 were labelled with [32P]orthophosphate and subjected to immunoprecipitation, SDS-PAGE, and autoradiography. Immunoblotting with monoclonal antibody B10 was used to control for equivalent levels of ER protein (Fig. E). A single band at 67 kDa in HEG0- but not in pSG5-transfected cells shows that hERα is phosphorylated in untreated cells (Fig. E, lanes 1 and 2). Cotransfection of pSG5-PKA resulted in a 5.6-fold increase in the intensity of the phosphorylated band (lane 3). 8-Br-cAMP treatment similarly increased hERα phosphorylation 3.65-fold (lane 6). HEG0236A was phosphorylated to a lower extent than was HEG0 (lane 4), and cotransfection of pSG5-PKA (lane 5) or the addition of 8-Br-cAMP (lane 7) resulted in lower increases (2.7- and 1.44-fold, respectively) in phosphorylation intensities compared to HEG0; 2.0- and 2.5-fold-lower signals were obtained for HEG0236A than for HEG0 with pSG5-PKA and 8-Br-cAMP, respectively.
Comparison of in vitro DNA binding by wild-type and mutant hERα.
Since serine 236 lies within the second zinc finger (CII) of the DBD (Fig. B) we wished to investigate the effect of its mutation on DNA binding by hERα. The mutants in which serine 236 was replaced by alanine in HEG0, HE15, and HEG19 were used to investigate DNA binding when phosphorylation at this position is prevented. Mutants in which serine 236 of HEG0, HE15, and HEG19 were replaced by glutamic acid were also created in order to examine the effect of a “constitutive” negative charge at this position. As PKA is a serine-threonine kinase, serine 236 was also mutated to threonine.
HEG0, HE15, and the serine 236 mutants were overexpressed in COS-1 cells by transient transfection; cells were then harvested, and extracts were prepared in a high salt buffer. Gel shifts were performed by preincubation of the extracts in gel shift buffer (containing 80 mM KCl) at 4°C for 15 min, followed by the addition of radiolabeled ERE and incubation at 25°C for 15 min, before the receptor-ERE complexes were analyzed by PAGE as previously described (59
). A specific retarded complex was observed for HEG0, and faster-migrating complexes were seen for HE15 and HEG19 (Fig. A, lanes 2, 6, and 10). Mutation of serine 236 to alanine or threonine had no obvious effect on complex formation (Fig. A, lanes 3, 5, 7, 9, 11, and 13). Interestingly, replacement of serine 236 with glutamic acid drastically reduced the amount of complex seen (Fig. A, lanes 4, 8, and 12). Immunoblotting showed that the reduced DNA binding by HEG0236E
, and HEG19236E
was not due to the absence of protein or to its degradation during incubation (Fig. A, lower panel). These results suggest that the phosphorylation of serine 236 may adversely affect DNA binding by hERα.
FIG. 3 Comparison of the in vitro DNA binding by wild-type hERα and mutants of serine 236. (A) COS-1 cells were transiently transfected with pSG5 (lane 1), HEG0 (lane 2), mutants of HEG0 (lanes 3 to 5), HE15 and its mutants (lanes 6 to 10), or HEG19 (more ...)
In order to investigate the effect of ligand on DNA binding by the wild-type and mutant ERα, COS-1 cells grown in DMEM lacking phenol red and containing 5% charcoal-stripped FCS were transfected with HEG0, HEG0236A, or HEG0236E, and ligands were added 1 h prior to harvesting. No differences in DNA binding were observed between HEG0 and HEG0236A in the absence of ligand or in the presence of 17β-estradiol (E2), the partial agonist 4-hydroxytamoxifen (OHT), or the complete antagonist ICI 182,780 (ICI) (Fig. B lanes 2 to 9). As described above (Fig. A), little binding was evident for HEG0236E in the absence of ligand (Fig. B, lane 10). In the presence of E2 and OHT, however, some DNA binding was seen (lanes 11 and 12), although no DNA binding was observed with ICI (lane 13), suggesting that the inhibition of DNA binding can be prevented, at least partially, by some ligands. Again, immunoblotting was used to control for levels of protein and degradation (Fig. B, lower panel).
In order to demonstrate that the apparent inhibition of DNA binding is due to phosphorylation of serine 236 and to evaluate its ligand dependence, cells were transfected with HEG0 or HEG0236A
, as well as the expression vector pSG5 containing the open reading frame encoding the catalytic subunit of PKA (pSG5-PKA) (73
). Gel shifts performed under standard conditions demonstrated that the overexpression of PKA had little effect on DNA binding by HEG0 in the presence of E2 or OHT (Fig. A, lanes 4 to 7). In the absence of ligand (Fig. A, lanes 2 and 3) or in the presence of ICI (Fig. A, lanes 8 and 9), however, in vitro DNA binding was dramatically reduced when HEG0 was cotransfected with pSG5-PKA. No reduction in DNA binding was observed when HEG0236A
was cotransfected with pSG5-PKA in the presence or absence of any of the ligands (Fig. A, lanes 10 to 17).
FIG. 4 DNA binding by hERα is inhibited by phosphorylation of serine 236. (A to D) COS-1 cells were transiently transfected with pSG5, HEG0, or HEG0236A. E2, OHT, and ICI were added 2 h prior to harvesting. (A and C) An expression plasmid encoding the (more ...)
Activation of endogenous PKA by 8-Br-cAMP resulted in a similar loss of DNA binding by HEG0 in the absence of ligand (Fig. B, compare lanes 2 and 6) or in the presence of ICI (Fig. B, compare lanes 5 and 9) but not in the presence of E2 or OHT (Fig. B, lanes 3, 4, 7, and 8). As seen when PKA was overexpressed, no differences in DNA binding were evident for HEG0236A in the absence or presence of 8-Br-cAMP (Fig. B, lanes 10 to 17). Similar results were obtained when forskolin was used (data not shown). Overexpression of the catalytic subunit of PKA or activation of PKA with 8-Br-cAMP did not result in altered protein levels in transfections, nor did they increase the degradation of wild-type or mutant ERα (Fig. A and B, lower panels).
H89 is a specific inhibitor of PKA (35
). Treatment with H89 prevented the loss of DNA binding by HEG0, a result observed in the absence of ligand when PKA was overexpressed (Fig. C, compare lanes 2 and 3 with lane 4). The loss of DNA binding observed in the presence of ICI was also prevented when H89 was added (Fig. C, compare lanes 8 and 9 with lane 10). The reduction in DNA binding induced by 8-Br-cAMP was similarly prevented by H89 (Fig. D). There was no effect of H89 on the DNA binding by HEG0 in the presence of E2 or OHT (Fig. C and D, lanes 5 to 8) or on the DNA binding by HEG0236A
in the absence of ligand or in the presence of E2, OHT, or ICI (Fig. C and D, lanes 11 to 20).
Replacement of serine 236 by glutamic acid inhibits dimerization by hERα.
ERα binds DNA as a homodimer or as a heterodimer with ERβ (63
). Inability of the receptor to dimerize would result in the loss of DNA binding. In order to investigate the effect of the mutation of serine 236 on dimerization, we analyzed the dimeric status of hERα bound in vitro to an ERE by using extracts of COS-1 cells transfected with HEG0 or HEG19 and cotransfected with the mutants of serine 236. The HEG19-ERE complex migrates faster than the complex obtained with HEG0 (Fig. A; Fig. A compare lanes 2 and 7). A weak lower band observed for HEG0 probably corresponds to a degradation product (see also Fig. and ) and has also previously been observed (60
). When coexpressed in COS-1 cells, an additional complex migrating at a position intermediate between those observed for HEG0 and HEG19 is observed, corresponding to the binding of HEG0-HEG19 heterodimers (Fig. A, lane 3). HEG0 and either HEG19236A
formed similar amounts of heterodimer as HEG0 and HEG19 (Fig. A, lanes 3 to 5). Heterodimer formation was similar when HEG0236A
were cotransfected with HEG19 (Fig. A, lanes 8 to 9). When HEG0 was cotransfected with HEG19236E
, however, a complex corresponding to HEG19236E
was not observed, nor was a heterodimeric complex seen while the HEG0 homodimeric complex was present at levels similar to those seen with HEG0 alone (Fig. A, compare lanes 2 and 6). The reciprocal event was observed when HEG0236E
was cotransfected with HEG19 (Fig. A, compare lanes 7 and 10). The fact that the amount of HEG0 and HEG19 complexes observed when cotransfected with HEG19236E
, respectively, is similar to levels observed for HEG0 and HEG19 alone suggests that dimerization is impaired by mutation of serine 236 to glutamic acid.
FIG. 5 Loss of hERα DNA binding is due to inhibition of dimerization. (A) Gel shifts were performed with extracts prepared from COS-1 cells transfected with HEG0 alone (lane 2) or together with wild-type HEG19 or serine 236 mutants (lanes 3 to 6) and (more ...)
The effect of mutation of serine 236 to glutamic acid upon dimerization was ascertained directly by using an assay in which HEG0, HEG19, and their respective glutamic acid mutants were synthesised by in vitro transcription followed by in vitro translation with a rabbit reticulocyte lysate system in the presence of 35S-labeled methionine, followed by immunoprecipitation of one-half of the lysates with the monoclonal antibody B10, which recognizes an epitope in the A/B region and does not therefore, recognize HEG19 (or its mutants) (Fig. A). Immunoprecipitation of HEG0 gave a band at 67 kDa, whereas HEG19 was not immunoprecipitated by B10 (Fig. B, compare lanes 1 and 3). Immunoprecipitation of HEG0 and HEG19 cotranslated with B10 resulted in the detection of HEG0 and HEG19, showing that they can dimerize. Only one band, at 67 kDa, was observed when HEG0236E and HEG19 (Fig. B, lane 5) or HEG0 and HEG19236E (Fig. B, lane 6) were cotranslated and immunoprecipitated with B10, indicating that the mutation of serine 236 to glutamic acid impairs dimerization by hERα. Immunoprecipitation of the second half of all of the lysates with F3, which recognizes an epitope present in both HEG0 and HEG19 (Fig. A), was also performed to show that HEG19 and HEG19236E were not absent in lysates in which they were not coprecipitated with HEG0236E (Fig. B, lanes 8 to 14).
We and others have previously shown that hERα and hERβ can heterodimerize (22
). Given the almost complete amino acid sequence identity between hERα and β in the DBD and their ability to heterodimerize, we investigated whether hERα and β can heterodimerize in solution and whether heterodimerization between hERα and β is impaired by mutation of serine 236 of hERα to glutamic acid. HEG0, HEG0236E
, and hERβ1 (pSG5 containing the hERβ open reading frame with the FLAG tag at the 5′ end, enabling immunodetection with the M2 monoclonal antibody [63
]) were in vitro translated in the presence of 35
S-labeled methionine, and immunoprecipitations were performed with B10 and M2 monoclonal antibodies as shown (Fig. C). Immunoprecipitation of HEG0 with hERβ1 by using B10 or M2 resulted in immunoprecipitation of both polypeptides (Fig. C, lanes 2 and 7), whereas when HEG0236E
and hERβ1 were immunoprecipitated with B10 only HEG0236E
was seen (Fig. C, lane 4). Immunoprecipitation with M2 resulted in the coprecipitation of HEG0 but not of HEG0236E
with hERβ1 (Fig. C), indicating that serine 236 is important for the dimerization of hERα and -β, as well as for homodimerization of hERα.
Phosphorylation on serine 236 inhibits dimerization by hERα in the absence of ligand.
E2 and OHT can overcome the inhibitory effect on DNA binding of mutating serine 236 to glutamic acid or activation of PKA (Fig. and ). The gel shift and immunoprecipitation results described above show that the 236E mutants fail to dimerize (Fig. ). In order to determine whether the inhibition of dimerization is overcome by ligand treatment, COS-1 cells were transiently transfected with HEG0 and HEG19 or their respective mutants, either separately or together. The cells were labelled with [35S]methionine, and WCEs were immunoprecipitated with B10. Immunoblotting of extracts with F3 established that HEG0 and/or HEG19 (and their mutants) were present in the appropriate extracts (Fig. B, lower panel). In order to investigate the effects of phosphorylation of serine 236, the cells were transfected with pSG5 or pSG5-PKA in addition to the ERα constructs.
FIG. 6 Inhibition of dimerization by PKA is prevented by 17β-estradiol and 4-hydroxytamoxifen but not by ICI 182,780. COS-1 cells were transiently transfected with HEG0, HEG19, HEG0236A, HEG19236A, HEG0236E, and HEG19236A and pSG5-PKA as shown. The cells (more ...)
Monoclonal antibody B10 immunoprecipitated HEG0, HEG0236A, and HEG0236E (Fig. A, lanes 2, 8, and 18, respectively) but not HEG19 or HEG19236A (lanes 3 and 9). As expected, immunoprecipitation of WCEs from cells transfected with HEG0 and HEG19 resulted in precipitation of HEG19 along with HEG0 in the presence or absence of E2 (Fig. A, lanes 4 and 5). When HEG0 and HEG19 were transfected along with pSG5-PKA, however, HEG19 was not brought down in the absence of ligand (Fig. A, lane 6). HEG19 was brought down when E2 was added in vivo (lane 7), indicating that phosphorylation of hERα by PKA inhibits dimerization in vivo in the absence but not in the presence of E2. In agreement with this, immunoprecipitation of WCEs containing HEG0236A, HEG19, and pSG5-PKA or containing HEG0236A, HEG19236A, and pSG5-PKA resulted in the coprecipitation of HEG19 and HEG19236A, respectively, in the presence or absence of E2 (Fig. A, lanes 10 to 17). Immunoprecipitation of WCEs from cells transfected with HEG0236E and HEG19 resulted in coprecipitation of HEG19 in the presence but not in the absence of E2 (Fig. A, lanes 19 and 20).
Similar experiments performed in the presence of OHT or ICI showed that the PKA-mediated inhibition of dimerization is prevented by OHT (Fig. B, lanes 2 and 4). OHT also enabled the dimerization between HEG0236E and HEG19 (lane 10). The addition of ICI, however, did not prevent inhibition of dimerization by PKA (Fig. B, lanes 3 and 5), nor did HEG0236E and HEG19 dimerize in the presence of ICI (lane 11).
The importance of ligands for heterodimerization of hERα and -β was also investigated. COS-1 cells were transiently transfected with HEG0, HEG0236A, or HEG0236E in the presence or absence of hERβ1 and pSG5-PKA and labeled with [35S]methionine. The lysates were divided into three equal fractions, and immunoprecipitations were performed with B10 (Fig. A) or M2 (Fig. B) monoclonal antibodies. As expected, hERβ1 was coimmunoprecipitated with HEG0 in the presence or absence of ligand (Fig. A, lanes 4 to 7). Overexpression of the catalytic subunit of PKA (pSG5-PKA), however, inhibited dimer formation in the absence of ligand (Fig. A, lane 8), and addition of ICI did not prevent the inhibition (Fig. A, lane 11), as found for hERα. Addition of E2 enabled heterodimer formation (Fig. A, lane 9). However, whereas the addition of OHT allowed dimerization between HEG0 and HEG19, dimer formation between HEG0 and hERβ1 in the presence of OHT was undetectable when the catalytic subunit of PKA was overexpressed (Fig. A, lane 10). Dimer formation between HEG0236A and hERβ1 was ligand independent and was not inhibited by PKA (Fig. A, lanes 13 to 20). Furthermore, in contrast to the observations with hERα, no heterodimer formation between HEG0236E and hERβ1 was seen in the presence of E2 or OHT (Fig. A, lanes 22 to 29). Immunoprecipitations performed with M2 to immunoprecipitate hERβ1 gave results similar to those obtained for HEG0 with B10 (Fig. B). Immunoblotting of the remainder of these lysates with B10 and M2 served to control for the presence of HEG0 (or mutants of serine 236) and hERβ1, respectively, in the appropriate lysates (Fig. C).
FIG. 7 Dimerization between hERα and -β is inhibited by PKA. COS-1 cell extracts were immunoprecipitated with B10 (A) or M2 (B) after transient transfection with HEG0, HEG0236A, HEG0236E, and/or hERβ1, as well as pSG5-PKA (as appropriate), (more ...) Stimulation of hERα-mediated transcriptional activation by PKA is modulated in a promoter-specific manner.
The results described above indicate that phosphorylation of serine 236 inhibits hERα dimerization in the absence of ligand or in the presence of ICI. In order to determine whether overexpression of PKA results in the prevention of transcriptional activation by hERα in a serine 236-dependent manner, we examined the ability of wild-type and mutant hERα to activate ERE-containing reporter genes. Previous studies have shown that stimulation of PKA results in a synergistic increase in transcriptional activation by hERα in the presence of E2 or OHT in a cell type-specific and promoter-independent manner, whereas PKA-induced transactivation by ERα in the absence of ligand is not observed in MCF7 cells (21
) and in other cell types is promoter context dependent (42
We examined transcriptional activation by HEG0, HEG0236A, and HEG0236E in COS-1 cells with a number of CAT-based reporters driven by minimal promoters containing one or three EREs (ERE-TATA-CAT and ERE-3-TATA-CAT) or more complex promoters comprising one or three EREs and the thymidine kinase (ERE-1-tk-CAT and ERE-3-tk-CAT) or the globin promoter (ERE-G-CAT) (Fig. ). HEG0 activated each promoter in the presence of E2 and to a lesser extent in the presence of the partial agonist OHT (Fig. A to F, lanes 2 to 5). Little transactivation was observed in the presence of ICI or in the absence of ligand. In the case of each reporter levels of activation were similar for HEG0236A and HEG0 (Fig. A to F, compare lanes 2 to 5 with lanes 10 to 13). Transactivation by HEG0236E was lower than that observed for HEG0, although transcriptional activation in the presence of E2 and OHT was evident in the presence of E2 ranging between 23% (Fig. A) and 60% (Fig. F) relative to HEG0. Similar differences in the levels of trans-activation were observed for HEG0236E compared to HEG0 in the presence of OHT (Fig. A to F, compare lanes 2 to 5 with lanes 18 to 21).
FIG. 8 Comparison of the transcriptional activities of hERα mutants on various estrogen-responsive reporter genes. (A to F) Transcriptional stimulation in COS-1 cells of six reporter genes by the wild-type human ERα (HEG0) and the serine 236 (more ...)
Cotransfection of HEG0 with pSG5-PKA resulted in a 1.6- to 14-fold increase (relative to the activity observed in the absence of PKA) in transactivation in the presence of E2 depending on the reporter gene used (Fig. A to F, compare lanes 3 and 7). PKA resulted in a 1.0- to 4.7-fold increase in OHT-induced transactivation by HEG0 (Fig. A to F, compare lanes 4 and 8). In the absence of ligand, cotransfection of pSG5-PKA did not yield any increase in transactivation in the case of the ERE-TATA-CAT, ERE-1-tk-CAT, ERE-3-tk-CAT, or the vit-tk-CAT reporter genes (Fig. A, C, D, and E), whereas 1.4- and 4.9-fold increases were observed for ERE-3-TATA-CAT and ERE-G-CAT (Fig. B and F), respectively. PKA also caused 1.5- and 2.2-fold increases in transactivation in the presence of the pure antiestrogen ICI in the case of ERE-3-TATA-CAT and ERE-G-CAT, respectively (Fig. B and F, compare lanes 5 and 9), but no stimulation was observed with the other reporter genes.
These results show that PKA synergizes with E2 and OHT to increase the transactivational ability of hERα. PKA-mediated transactivation in the absence of ligand is promoter dependent and, furthermore, PKA can also stimulate transactivation by hERα in the presence of the “pure” antiestrogen ICI, again in a promoter-specific manner. Similarly, in MCF7 cells transfection of PKA or the addition of 8-Br-cAMP resulted in increased transactivation in the presence of E2 and OHT from all of the reporter genes described above. However, no increase in transactivation in the presence of ICI or the absence of ligand was observed with any reporter gene, thus confirming previous reports indicating that ligand-independent transactivation mediated by PKA is cell type specific (data not shown) (9
For HEG0236A, PKA-mediated increases in transactivation were similar to those observed for HEG0 in the presence of E2 and OHT (Fig. A to F, compare lanes 11 and 15 and lanes 12 and 16). Whereas PKA stimulated transactivation by HEG0 in the absence of ligand only when the ERE-G-CAT reporter was used, in the case of HEG0236A, increased transactivation by PKA was evident for all reporter genes (3.2- to 4.8-fold) (Fig. A to E, compare lanes 10 and 14). Similar increases were observed in the presence of ICI (Fig. A to F, lanes 13 and 17).
PKA also increased transactivation by HEG0236E in the presence of E2 and to a lesser extent in the presence of OHT (Fig. A to F, compare lanes 19 and 20 with lanes 23 and 24). As expected, no increase in transactivation was observed in the presence of ICI or in the absence of ligand, except that ERE-G-CAT (which gave a ligand-independent increase in transactivation of HEG0 by PKA) was stimulated to a similar extent (3.2-fold) in the absence of ligand (compare lanes 18 and 22) and in the presence of E2 or OHT (Fig. F, compare lanes 19 and 20 and lanes 23 and 24). Some increase (twofold) was also obtained in the presence of ICI (Fig. F, compare lanes 21 and 25).
Collectively, the above data indicate that phosphorylation of serine 236 does indeed inhibit the transcriptional activation by hERα in the absence of E2 or OHT. In order to directly show that PKA can inhibit DNA binding by hERα in vivo, we exploited a transcriptional interference assay which has previously been used to show that hERα can bind to an ERE in vivo in the presence of ICI (60
). We used a reporter gene containing a binding site for the yeast transcriptional activator GAL4 (17M), an ERE, and the adenovirus major late promoter TATA box driving the CAT gene (79
). Cotransfection of this reporter gene (17M-ERE-TATA-CAT) and GAL-VP16, in which the DBD (amino acids 1 to 147) of GAL4 is fused to the transcription activation function of the herpes simplex virus VP16 resulted in transcriptional activation (taken as 100%), which was unaffected by the coexpression of PKA (Fig. G lanes 2 and 3). Cotransfection of HEG0 and GAL-VP16 resulted in a 4-fold increase in CAT gene expression in the presence of E2 (lane 9) and a 1.8-fold increase in the presence of OHT (lane 10). In the presence of ICI, however, a fourfold decrease in expression was observed, a finding indicative of repression due to the specific DNA binding of IC-bound HEG0 (lane 11). It is not clear why the inhibition of expression is not seen in the absence of ligand (lane 8), but it may be a reflection of residual estrogens in the culture medium (see reference 60
). Cotransfection of pSG5-PKA resulted in a further increase in transactivation in the presence of E2 and OHT but a repression of expression in the presence of ICI was lost (lane 15), thus showing that PKA inhibits DNA binding by HEG0 in the presence of ICI.
In the case of HEG0236A, ICI inhibited expression even in the presence of PKA (lane 27), indicating that DNA binding by HEG0236A is not inhibited by PKA. HEG0236E cotransfection with GAL-VP16 had little effect on expression in the absence or presence of PKA, suggesting that there is little DNA binding by HEG0236E (Fig. G, lanes 32 to 39), a finding in agreement with the results presented above.