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by Cdk7-mediated phosphorylation
1 The Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan
2 SORST, Japan Science and Technology, Kawaguchi, Saitama, Japan
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Abstract |
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(hER) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ER ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hER transactivation in vitro through Ser118 phosphorylation. To investigate an impact of Cdk7 on hER transactivation in vivo, we assessed activity of hER in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hER in the eye disc. Ectopic expression of the wild-type or mutant receptors, together with a green fluorescent protein (GFP) reporter gene, allowed us to demonstrate that hER expressed in the fly tissues was transcriptionally functional and adequately responded to hER ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser118 with alanine in hER (S118A mutant) significantly reduced the ligand-induced hER transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hER exhibited reduced response to the ligand; levels of transactivation by the hER S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hER at Ser118 has been observed in vitro by both human and Drosophila Cdk7. Our findings demonstrate that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER
in vivo via Ser118 phosphorylation. |
Introduction |
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and ß, belong to the nuclear receptor superfamily and act as ligand-induced transcription factors. As in other nuclear receptor superfamily members, structure of ER proteins is divided into five or six functional domains (designated as A to E/F domains). The highly conserved DNA binding domain is located in the C domain, while the ligand-binding domain (LBD) is mapped to the E/F domain. Transactivation function is present in the N-terminal A/B domain (AF-1) and in the C-terminal LBD (AF-2) (Kumar et al. 1987; Tora et al. 1989). Although both AF-1 and AF-2 are involved in the ligand-dependent transactivation function of ERs, AF-1 is constitutively active, while AF-2 activity is dependent on ligand binding (Endoh et al. 1999; Kobayashi et al. 2000; Watanabe et al. 2001). AF-1 and AF-2 domains have distinctive properties and their activities may depend on cell type and promoter context (Kumar et al. 1987; Tora et al. 1989). ER target gene promoters contain oestrogen-response elements (EREs) that are recognized and directly bound by ER homo- or hetero-dimers followed by chromatin remodelling, presumably by recruited ATP-dependent chromatin remodelling complexes (Belandia & Parker 2003; Kitagawa et al. 2003). ERE-bound liganded ERs also induce recruitment of a number of histone acetyltransferase (HAT) and non-HAT cofactors that activate transcription (McKenna & O'Malley 2002). HAT coactivator complexes, CBP/p160 (Onate et al. 1995; Kamei et al. 1996; Chen et al. 1997; Spencer et al. 1997) and TRRAP/GCN5 (Yanagisawa et al. 2002), and non-HAT DRIP/TRAP complexes (Fondell et al. 1996; Yuan et al. 1998; Naar et al. 1999; Rachez et al. 1999) are thought to act as common coactivator complexes for ERs as well as for other DNA-binding transcription factors. Therefore, ligand binding leads to structural alteration and switch of ER function from transcriptional repression to transcriptional activation via the recruitment of coactivators (Shiau et al. 1998; Freedman 1999; Glass & Rosenfeld 2000; Metivier et al. 2003).
It is well known that phosphorylation of ER modulates the activity of both AF-1 and AF-2 (Ali et al. 1993; Le et al. 1994; Kato et al. 1995; Chen et al. 2000). Among sites of potential phosphorylation, Ser118 residue (S118) in the hER AF-1 domain has been particularly intensively studied with regard to the state of its phosphorylation and consequent potentiation of AF-1 activity. We have previously demonstrated that Ser118 is phosphorylated by ERK, a MAPK activated by the epidermal growth factor (EGF) or insulin-like growth factor (IGF) signalling, that results in the AF-1 potentiation in cultured cells (Kato et al. 1995). More recently, Chen and colleagues have shown that Cdk7 also phosphorylates hER Ser118 in an oestrogen-dependent manner and enhances ER transactivation in mammalian cells in culture (Chen et al. 2000). As Cdk7 is a key subunit of the basal transcription factor TFIIH complex (Frit et al. 1999; Egly 2001), it has been suggested that this phosphorylation takes place when TFIIH is recruited adjacent to hER, presumably in the transcription initiation complex. Therefore, accumulating evidence suggests that phosphorylation of hER Ser118 may play a significant role in regulation of AF-1 activity. However, the physiological role of Ser118 phosphorylation and associated kinases in hER function remain to be established in vivo.
In Drosophila melanogaster, at least 20 members of the nuclear receptor (NR) family, such as the ecdysone receptor (EcR), have been genetically identified that, similar to the vertebrate NRs, are thought to transcriptionally control expression of target genes (Talbot et al. 1993; Baker et al. 2003). Recently, we reported that human androgen receptor ectopically expressed in Drosophila tissues was transcriptionally active and responsive to AR agonists and antagonists (Takeyama et al. 2002). In the present study, to assess an impact of Ser118 phosphorylation by Cdk7 and related kinases on hER activity in vivo, we generated transgenic Drosophila lines in which hER was ectopically expressed in specific Drosophila tissues using a GAL4/UAS system (Brand & Perrimon 1993). hER expressed in the fly was transcriptionally functional and responded adequately to ER ligands, as expected from mammalian studies. Apparently, for its transactivation function in these transgenic flies, hER recruited endogenous co-activators, such as those shown to be homologous to mammalian CBP and AIB1 (Akimaru et al. 1997; Bai et al. 2000). We found that replacement of S118 with alanine residue (S118A) in hER resulted in the marked reduction of ligand-induced hER transactivation in transgenic fly eye disc. Furthermore, in a cdk7 inactive mutant Drosophila (cdk7ts
) (Larochelle et al. 2001), transactivation by the wild-type but not the S118A hER was significantly reduced. In addition, both human and Drosophila recombinant Cdk7 were equally able to phosphorylate hER at Ser118
in vitro. We have also shown that Cdk7 acts as a co-activator of hER transactivation in transfected cells in culture. Therefore, our results provide for the first time genetic evidence that phosphorylation of Ser118 potentiates transcriptional activity of hER and that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER
in vivo through Ser118 phosphorylation.
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Results |
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in Drosophila is transcriptionally functional
Our previous studies showed that human androgen receptor ectopically expressed in Drosophila tissues was adequately functional (Takeyama et al. 2002). We have utilized the same strategy to generate transgenic Drosophila expressing hER together with ERE-dependent green fluorescent protein (GFP) as a reporter gene. Wild-type hER (HEG0), AF-1 (HE15) or AF-2 (HE19) domains (as illustrated in Fig. 1A) were ectopically expressed in photoreceptor cells under control of the glass multimer reporter (GMR) gene promoter (Moses & Rubin 1991) using the Drosophila melanogaster GAL4-UAS system (Brand & Perrimon 1993). The eye disc, one of several larval discs in Drosophila, has been shown to be an effective model to assess Cdk7 function as a cell survival signal. Expression of hER proteins was estimated by staining with immunofluorescent antibody. Levels of GFP reporter expression in respective eye discs were quantified by green fluorescence and normalized against the levels of ER protein to determine fold of activation.
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AF-1 and AF-2 activities were observed in Schneider (S2) cells derived from Drosophila embryos (Fig. 1C). These data indicated that hER ectopically expressed in Drosophila tissues was adequately functional in ligand-induced transactivation, presumably through recruitment of endogenous co-regulators. Therefore, it appears that human steroid receptors ectopically expressed in Drosophila retain their transactivation function.
Co-activation of hER by Drosophila CBP and p160 HAT homologues
As hER was transcriptionally functional in insect cells in culture and in Drosophila eye disc cells in vivo, ability of endogenous fly co-activators to modulate hER transactivation was assessed in mutant flies deficient for Drosophila homologues of mammalian p160 (tai) or CBP (nej) (Akimaru et al. 1997; Bai et al. 2000). The oestrogen-induced transactivation function of hER was clearly reduced in both of these mutants without affecting levels of hER expression (Fig. 2). These data suggest that Drosophila homologue of the mammalian p160/CBP HAT complex acts as a co-activator of hER in the fly cells. This was further confirmed by the observation of enhanced hER transactivation in flies over-expressing TAI, Drosophila AIB1 homologue, in the eye disc.
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AF-2 via the direct association of p160 family member proteins with helix 12 of the hER LBD (Onate et al. 1995; Chen et al. 1997; Heery et al. 1997). However, little is known about the role of the p160/CBP complex in modulation of hER AF-1 activity. Although it is presumed that the complex bridges the AF-1 and AF-2 domains to synergistically enhance hER transactivation function (Kobayashi et al. 2000), the p160/CBP complex was also able to enhance transcriptional activity of the AF-1 domain alone (i.e. the HE15 mutant). Indeed, similar patterns of AF-1 domain (HE15) and full-length hER (HEG0) transactivation in mutant flies (Fig. 2) suggest that hER AF-1 activity is modulated in vivo by the p160/CBP co-activator complex.
Significant role of Ser118 in hER function in vivo
In mammalian cells, the potentiation of hER AF-1 by phosphorylation of the Ser118 residue has been well documented (Kato et al. 1995; Chen et al. 2000). However, the impact of Ser118 phosphorylation in hER transactivation function has not yet been verified in vivo. We tested the significance of hER Ser118 in the insect S2 cells transfected with hER mutants containing a serine to alanine replacement at position 118 (HE457, HE15/457) (Fig. 3A and 3B). These mutants exhibited decreased transactivation capacities even though levels of the mutant expression appeared to be similar to that of wild-type hER.
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function in transgenic flies (Fig. 3C). Although mutant and wild-type hER expression levels in third instar larval eye discs were indistinguishable, a clear reduction in GFP induction was observed in the alanine replacement mutants. These findings provided evidence that the Ser118 residue played a pivotal role in hER transactivation in vivo.
In vivo potentiation of hER by Cdk7-mediated phosphorylation at Ser118
As it is likely that the Ser118 residue could be phosphorylated by a number of endogenous protein kinases to support hER transactivation, we studied the ability of dCdk7 to phosphorylate hER at Ser118
in vitro and in vivo. The serine/threonine kinase Cdk7 is indispensable for transcription initiation by RNA polymerase II as an essential component of the transcription factor TFIIH complex (Frit et al. 1999; Egly 2001). dcdk7ts
mutant flies express a temperature-sensitive Cdk7 mutant that is inactive at temperatures at or above 30 °C (Larochelle et al. 2001). We assessed transactivation function of HEG0 and HE457 in these dcdk7ts
mutant flies (Fig. 4, left panel). Oestrogen-induced transactivation of HEG0 in dcdk7ts
flies was significantly reduced at 30 °C in comparison with that at room temperature (25 °C). In contrast, HE457 transactivation in dcdk7ts
flies was not affected by exposure to high temperatures (Fig. 4, right panel). These results indicate that Cdk7 potentiated hER transactivation in vivo through Ser118 phosphorylation.
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Ser118 is a substrate for dCdk7 in vitro. A recombinant GST-fused hER segment (amino acids 56–180) chimera protein expressed in E. coli, and dCdk7 and hCdk7 expressed in 293T cells were used for the in vitro phosphorylation assay (Fig. 5A). GST-fused human retinoic acid receptor 1 (hRAR1), a well-characterized substrate for the mammalian Cdk7 (Rochette-Egly et al. 1997) was used as a positive control. dCdk7 and hCdk7 were equally capable of phosphorylating hER and hRAR1. However, the Cdk7 phosphorylation was clearly reduced when the S118A mutant (HE457) was used as a substrate (Fig. 5B).
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S118A mutant by Drosophila AIB1 homologue
Finally, using a fly line with ectopical over-expression of Drosophila AIB1 homologue (TAI) in the eye disc, we addressed a question whether enhancement of hER transactivation by the p160/CBP complex is dependent on the receptor Ser118 phosphorylation status. Although the hER S118A mutant was less effective in the ligand-induced transactivation, TAI significantly enhanced transcriptional activity of both the mutant and the wild-type receptor (Fig. 6). This suggests that modulation of the ligand-induced hER transactivation by the p160/CBP co-activator complex does not depend on the receptor phosphorylation.
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Discussion |
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expressed in Drosophila is functional in ligand-induced transactivation
We have previously shown that the human androgen receptor (hAR) ectopically expressed in Drosophila tissues was transcriptionally functional and responsive to hAR ligands similar to that in mammalian cultured cells and intact tissues (Takeyama et al. 2002). In the present study, we utilized the same approach and demonstrated that hER expressed in Drosophila was able to activate the ERE reporter gene and respond to hER agonists and antagonists in the same manner that had been observed in mammalian cells and tissues (McDonnell et al. 1995; Metzger et al. 1995; Watanabe et al. 2001). As hER transgenic flies appear to be normal in terms of growth and reproduction, without any overt abnormalities, it seems that human steroid hormone receptors do not significantly interfere with endogenous signalling pathways. It can also be inferred that exogenous human steroid receptors do not compete with endogenous NRs at the fly NR-responsive elements in target gene promoters (Talbot et al. 1993; McKenna & O'Malley 2002). Therefore, our results provided evidence that transgenic Drosophila expressing hER represent a potent and functionally relevant system in which to evaluate NR synthetic ligands and to genetically identify and characterize novel NR co-regulators.
Pivotal role of Ser118 in the hER ligand-induced transactivation function in vivo
Both N-terminal AF-1 and C-terminal AF-2 domains contribute to the hER ligand-induced transactivation function, with each AF-1 and AF-2 activity dependent on promoter-context and cell type (Kumar et al. 1987; Tora et al. 1989). The balance between hER AF-1 and AF-2 is thought to be responsible, at least in part, for the tissue-specific action of selective oestrogen receptor modulators (SERMs) such as tamoxifen (Berry et al. 1990; McDonnell et al. 1995; Metzger et al. 1995; Brzozowski et al. 1997; Shiau et al. 1998). In particular, the activity of hER AF-1 is believed to support the oestrogenic actions of SERMs (Endoh et al. 1999; Watanabe et al. 2001), leading to beneficial actions of SERMs in certain tissues such as the improved bone properties in oestrogen-related pathophysiological states (Shang & Brown 2002). Therefore, while the physiological and pharmacological significance of hER AF-1 activity has been well addressed, the molecular basis underlying AF-1 function remains to be elucidated in terms of identifying the relevant specific co-regulators and co-regulator complexes (Endoh et al. 1999; Watanabe et al. 2001). The core activation region of hER AF-1 has been mapped to the middle of the A/B domain (Kobayashi et al. 2000), and a number of in vitro studies have indicated that the Ser118 residue in this core region appears to play a crucial role and can be phosphorylated by several kinases in response to extracellular signals (Kato et al. 1995; Chen et al. 2000). Nevertheless, the impact of Ser118 phosphorylation in vivo remains obscure because of lack of studies involving intact animals. The present findings provide for the first time in vivo evidence for the significance of Ser118 phosphorylation in the transcriptional activity of the AF-1 domain alone and in the transactivation function of hER as a whole receptor.
In vivo potentiation of hER AF-1 through Cdk7-mediated phosphorylation of Ser118
It has been shown that hER Ser118 can be phosphorylated by several kinases (Ali et al. 1993; Le et al. 1994; Kato et al. 1995; Chen et al. 2000). Cdk7 has been chosen for the present study as mutant flies with inactive Cdk7 appear to suffer more general defects in gene regulation (Austin & Biggin 1996). We have shown that Cdk7 phosphorylates hER Ser118
in vivo and that this phosphorylation enhanced hER AF-1 activity in normal flies. It has been shown recently that, besides direct receptor phosphorylation, MAPKs are also able to potentiate function of some hER co-activators, including AIB1, through phosphorylation of the cofactor protein (Font de Mora & Brown 2000). This suggests an additional mechanism for downstream cross-talk between different signalling pathways. Our transgenic Drosophila provides an experimental system in which to further study whether MAPKs activated by growth factors or stress-induced signalling pathways can also modulate hER activity.
Ser118 phosphorylation-dependent and -independent co-activators for hER
The S118A hER mutant retained ligand responsiveness, albeit with reduced transactivation. Transactivation in the S118A hER mutant has nevertheless been significantly enhanced by over-expression of TAI, Drosophila AIB1 homologue. Therefore, it appears that hER activity is modulated in vivo by both phosphorylation-dependent and phosphorylation-independent co-activators. However, the timing of the recruitment of these co-activators, presumably within co-factor complexes associated with the AF-1 domain, remains unclear. p68/p72 have been identified as hER AF-1-specific co-activators that physically associate with the hER AF-1 domain (Endoh et al. 1999; Watanabe et al. 2001). Significantly, this interaction was clearly not dependent on Ser118 phosphorylation. It is not clear, however, whether recruitment of most of known hER co-activators is dependent on phosphorylation status of the receptor. In this respect, the transgenic Drosophila lines that express hER and its mutants represent a powerful tool for genetic screening of phosphorylation-dependent and -independent co-factors.
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Experimental procedures |
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hER mutants and dCdk7 expression vectors were constructed using the pCaSpeR vector for expression in Schneider cells. hER mutants and dCdk7 expression plasmids (0.05 µg) were co-transfected with 0.2 µg actin-GAL4 plasmid and 0.5 µg ERE-tk-luc plasmid, along with 10 ng pRL-CMV-luc plasmid as an internal control. Three hours after transfection, the ligands 10–8 M 17ß-oestradiol (Sigma, St Louis, MO), 10–8 M tamoxifen (Sigma) or 10–8 M ICI 182.780 (Tocris Cookson, Ballwin, MO) were added. After 20 h, dual luciferase assays were performed as previously described (Yanagisawa et al. 2002).
Generation of transgenic flies and Drosophila stocks
For germ-line transformation into Drosophila, cDNA encoding hER mutants and GFP reporter under control of ERE-containing promoter were inserted into pCaSpeR. Transgenic constructs together with p
25.7wc transposase were microinjected into w1118 embryos using a micromanipulator (Leica). Several independent transformant lines were established. To express hER in Drosophila eyes, transgenic lines were crossed with a GMR-GAL4 line that expressed GAL4 in the retina under the control of the glass multimer reporter. The taik05809
, UAS-tai, Df(1)J8254-Pw+[snf+, dhd+] and cdk7ts
mutants were obtained from the Bloomington Drosophila Stock Center. The nej3
and GMR-GAL4 line were the generous gifts of Drs S. Ishii and Y. Hiromi, respectively.
Histology
Eye imaginal discs from third instar larvae were dissected and fixed for 20 min in 4% formaldehyde at 25 °C. Eye discs were incubated with primary antibodies HC-20 (Santa Cruz Biotechnology, Santa Cruz, CA) or B10 that recognize the C- and N-terminal regions of hER, respectively. Cy5-conjugated Affinity Pure donkey anti-rabbit or anti-mouse IgG (Jackson Immuno-Research, West Grove, PA) were used as secondary antibodies for immunofluorescence staining. hER and GFP expression were detected using a Zeiss Confocal Laser Scanning System 510.
Western blotting
To confirm hER and GFP expression in Drosophila, cell lysates from the heads of adult flies of third instar larvae were separated by 15% SDS–PAGE and detected with anti-ER antibodies (HC-20 or B10) and anti-GFP antibody (Santa Cruz Biotechnology), and expression levels measured using Adobe Photoshop software facility. Fold-activation of hER in Drosophila was shown as GFP expression signal intensity normalizing with hER expression signal intensity.
In vitro phosphorylation
293T cells were transfected with FLAG tagged dCdk7 expression plasmid, lysed in lysis buffer, and immunoprecipitated with anti-FLAG affinity gel (Sigma). hCdk7 was obtained from 293T cells by immunoprecipitation with Cdk7 (N-19) antibody (Santa Cruz Biotechnology). dCdk7 or hCdk7 (9 µg) were incubated for 20 min at 30 °C with purified bacterially produced 10 µg of GST-fused hER (amino acids 56–180 of hER) and its mutants or GST-fused human retinoic acid receptor 1 (hRAR1) (Rochette-Egly et al. 1997), in 50 mM Tris-HCl, 0.5 mM EDTA, 25 mM MgCl2, 1 mM DTT, 20 µM ATP, 0.01 µCi [
-32P]ATP and 10% glycerol. Phosphorylation of substrates was analysed by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Expression of GST-hER mutants and GST-hRAR1 were detected by CBB staining.
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Acknowledgements |
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expression vectors and anti-hER antibody (B10). This work was supported by a grant-in-aid for priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (K.T. and S.K.) and Basic Research Activities for Innovative Biosciences (BRAIN) (S.K.). |
Footnotes |
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* Correspondence: Email: uskato{at}mail.ecc.u-tokyo.ac.jp
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Received: 27 May 2004
Accepted: 12 July 2004
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