In vivo potentiation of human oestrogen receptor alpha by Cdk7-mediated phosphorylation.
ABSTRACT Phosphorylation of the Ser(118) residue in the N-terminal A/B domain of the human oestrogen receptor alpha (hERalpha) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ERalpha ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hERalpha transactivation in vitro through Ser(118) phosphorylation. To investigate an impact of Cdk7 on hERalpha transactivation in vivo, we assessed activity of hERalpha in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hERalpha 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 hERalpha expressed in the fly tissues was transcriptionally functional and adequately responded to hERalpha ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser(118) with alanine in hERalpha (S118A mutant) significantly reduced the ligand-induced hERalpha transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hERalpha exhibited reduced response to the ligand; levels of transactivation by the hERalpha S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hERalpha at Ser(118) 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 hERalphain vivo via Ser(118) phosphorylation.
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ABSTRACT: The BTB domain is a highly conserved protein-protein interaction motif and functions in diverse cellular processes, including transcriptional regulation, ion channel assembly, cytoskeleton dynamics and apoptosis. Recently, it was reported that some BTB domain-containing proteins associate with Cullin-3 (Cul3), an E3 ubiquitin ligase, and act as an adaptor for Cul3 recognition of its substrate. However, the target substrates for the Cul3/BTB protein E3 ubiquitin ligase complex are largely unknown. Here, we report the characterization of a novel Drosophila BTB protein, dKLHL18/CG3571. By purification of a dKLHL18-associated complex, we identified CG10324, CG5808, l(2)37Cb and dCul3/guftagu. Indeed, the physical association of dKLHL18 with these proteins was observed in insect S2 cells, and genetic interactions among the identified factors were also observed in thorax development. Moreover, transient overexpression of dKLHL18 increased the ubiquitinated protein levels of CG10324 and CG5808. These findings suggest that dKLHL18 is an adaptor for a dCul3 E3 ubiquitin ligase to accommodate CG10324, CG5808 and l(2)37Cb proteins for ubiquitination.Genes to Cells 08/2009; 14(8):965-73. · 2.73 Impact Factor
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ABSTRACT: Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.Molecular and cellular biology 01/2009; 29(4):1017-34. · 6.06 Impact Factor
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ABSTRACT: PURPOSE: The oestrogen receptor (ERα) may be activated in a ligand-dependent manner, via oestrogen, or in a ligand-independent manner, via signal transduction pathways. Mitogen Activated Protein Kinase (MAPK) is known to directly phosphorylate ERα at serine 118 in a ligand-independent manner. This study investigated the interaction between MAPK and ERα in breast cancer. MATERIALS & METHODS: Immunohistochemical experiments were undertaken to determine the expression of MAPK, pMAPK and pER(ser118) in breast tumours to determine their clinical relevance. Immunofluorescent experiments were performed, on MCF-7 breast cancer cells, to monitor the phosphorylation and localisation of MAPK and ERα in response to oestrogen, heregulin and a MAPK inhibitor. RESULTS: Oestrogen and Heregulin stimulated phosphorylation of ERα and its nuclear translocation, but heregulin induced this at levels much lower than those observed with oestrogen. Following stimulation with heregulin, but not oestrogen, treatment with MAPK inhibitor reduced the levels of nuclear pER(ser118). In cells treated with both oestrogen and heregulin, nuclear pER(ser118) was visible; but at levels comparable with heregulin treatment alone. CONCLUSION: This study confirms that ligand-mediated phosphorylation is associated with rapid nuclear localisation of ERα, due to oestrogen binding. ERα is phosphorylated at serine 118 in a ligand-independent manner. Preventing nuclear translocation of pMAPK reduced the levels of ligand-independent, but not ligand-dependent phosphorylation of ERα. Co-stimulation with both oestrogen and heregulin suggested that heregulin mediated signalling determines the subcellular localisation of ERα. Activation of ERα by direct phosphorylation may result in its rapid deactivation due to degradation or nuclear export.European journal of cancer (Oxford, England: 1990) 12/2012; · 4.12 Impact Factor
Retraction: In vivo potentiation of human oestrogen
receptor a by Cdk7-mediated phosphorylation
Saya Ito, Ken-ichi Takeyama, Ayako Yamamoto, Shun Sawatsubashi, Yuko Shirode,
Alexander Kouzmenko, Tetsuya Tabata and Shigeaki Kato
Retraction: The above article in Genes to Cells (doi: 10.1111/j.1365-2443.2004.00777.x), pub-
lished online on 6 October 2004 in Wiley Online Library (http://onlinelibrary.wiley.com/),
has been retracted by agreement between the authors, the journal Editor in Chief, Mitsuhiro
Yanagida, and Wiley Publishing Asia Pty Ltd. The retraction has been agreed to due to multi-
ple usage of the tissue images in Figures 1(B), 2, 3(C), 4 and 6, and the absence of the gel
image for the lane 5 of ‘GFP’ in Figure 1(B).
Ito, S., Takeyama, K., Yamamoto, A., Sawatsubashi, S., Shirode, Y., Kouzmenko, A., Tabata, T. & Kato, S. (2004) In vivo poten-
tiation of human oestrogen receptor a by Cdk7-mediated phosphorylation. Genes Cells 9, 983–992. doi: 10.1111/j.1365-2443.
© 2013 The Authors
Genes to Cells (2013) 18, 1144
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd