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. 2004 Aug 9;32(14):4194-204.
doi: 10.1093/nar/gkh741. Print 2004.

TIP27: a novel repressor of the nuclear orphan receptor TAK1/TR4

Takeshi Nakajima  1 , Sanae Fujino, Gen Nakanishi, Yong-Sik Kim, Anton M Jetten
Affiliations

TIP27: a novel repressor of the nuclear orphan receptor TAK1/TR4

Takeshi Nakajima et al. Nucleic Acids Res. .

Abstract

The nuclear orphan receptor TAK1/TR4 functions as a positive as well as a negative regulator of transcription; however, little is known about the factors regulating or mediating its activity. Yeast two-hybrid analysis using the ligand-binding domain (LBD) of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27, which functions as a repressor of TAK1-mediated transactivation. TIP27 is a 27 kDa protein containing two zinc finger motifs. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. The region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1 while the TAK1-LBD from helix 3 until the C-terminus is required for the optimal interaction with TIP27. Pull-down assays demonstrated that the TIP27 physically interacts with TAK1 and supported the critical importance of the TID. Confocal microscopy showed that in the nucleus, TIP27 and TAK1 co-localize. TIP27 acts as a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve the inhibition of TAK1 homodimerization or DR1 binding but may be due to an effect on co-activator recruitment by TAK1. Our results indicate that TIP27 functions as a TAK1-selective repressor.

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Figures

Figure 1
Figure 1
(A) The amino acid sequence of human TIP27. The two zinc finger motifs (ZF1 and ZF2) and TID are indicated. The Cys and His residues participating in the tetrahedral configuration of the zinc fingers are indicated in boldface and underlined. The nuclear localization signal is boxed. The Glu/Asp-rich region is underlined. The TIP27 sequence was submitted to GenBank under accession no. AY372319. Bars indicate putative α-helical regions within TIP27. The consensus sequence of the two zinc finger motifs is shown at the bottom. (B) Partial sequence homology between TIP27 and a hypothetical Drosophila protein (DHP), the putative Drosophila homolog of TIP27. Zinc finger motifs are underlined.
Figure 2
Figure 2
Tissue-specific expression of TIP27. Poly(A)+ RNA (1.5 μg) from 10 different human tissues was examined by northern-blot analysis. Membranes were hybridized to a 32P-labeled probe for TIP27 as described in Materials and Methods.
Figure 3
Figure 3
TIP27 interacts specifically with the nuclear orphan receptor TAK1. CV-1 cells grown in DMEM containing charcoal-stripped FBS were transfected with (UAS)5-LUC, pRL-SV40, pM or pM-TIP27, and either VP16, VP16-TAK1, VP16-AR, VP16-ERα, VP16-PR-A, VP16-LXRα, VP16-PPARγ, VP16-RXRα, VP16-RTR or VP16-RORγ expression vectors as indicated. After 24 h, cells were treated with the respective ligand [1 μM 17β-estradiol (ERα), 22R-hydroxycholesterol (LXRα), GW347845 (PPARγ), SRI 11246 (RXRα), 5α-dihydrotestosterone (AR) or 100 nM progesterone (PR-A)] or vehicle and 48 h later assayed for reporter activities as described in Materials and Methods. The relative firefly LUC reporter activity was calculated and plotted as stated in Materials and Methods.
Figure 4
Figure 4
The TAK1 LBD is required for the interaction of TAK1 with TIP27. To determine the effects of various N- and C-terminal deletions and the I576N point mutation on the interaction of TAK1 with TIP27, CV-1 cells were transfected with (UAS)5-LUC, pRL-SV40, pM or pM-TIP27, VP16 or various VP16-TAK1 mutants, as indicated. After 48 h, cells were collected and assayed for reporter activities as described in Materials and Methods. The relative reporter activity was calculated and plotted.
Figure 5
Figure 5
Identification of TID in TIP27. (A) Effect of several TIP27 N- and C-terminal deletions on the interaction of TIP27 with TAK1. (B) Mapping of the TID: effect of various deletion and point mutations within the TID on the interaction of TIP27 with TAK1. CV-1 cells were transfected with (UAS)5-LUC, pRL-SV40, VP16 or VP16-TAK1, pM or various pM-TIP27 mutants, as indicated. After 48 h, the cells were collected and assayed for reporter activities as described in Materials and Methods. The relative reporter activity was calculated and plotted. The TID is localized between Asp39 and Lys79 of TIP27 (indicated by the hatched boxes). ZF, zinc finger domain.
Figure 6
Figure 6
TIP27 interacts directly with TAK1. (A) Schematic representation of GST-TIP27 fusion proteins used in pull-down assays. (B) and (C) GST and different GST-TIP27 fusion proteins were bound to glutathione–Sepharose 4B beads and then incubated with [35S]methionine TAK1 or the nuclear receptor RTR. After 1 h incubation, beads were washed extensively and bound proteins solubilized and analyzed by western-blot analysis. Radiolabeled proteins were visualized by autoradiography. (D) TAK1 pulls down TIP27, but not TIP27(ΔTID), from cellular extracts prepared from COS-1 cells co-transfected with p3XFlagCMV-TAK1 and pCMV-myc-TIP27 or pCMV-myc-TIP27(ΔTID). Protein lysates were prepared 48 h after transfection as described in Materials and Methods. One part was used in western-blot (WB) analysis using anti-Flag or anti-Myc antibodies, the remaining was used in IP assay using anti-Flag M2 agarose resin. Bound proteins were then examined by western-blot analysis using an anti-Myc antibody.
Figure 7
Figure 7
Localization of TIP27 and co-localization with TAK1. COS-1 cells were transfected with pEGFP-TIP27 (A) or co-transfected with pEGFP-TIP27 and p3XFlag-CMV-TAK1 (B–E) expression vectors as indicated. After 24 h, expression of EGFP-TIP27 and Flag-TAK1 fusion proteins was visualized using fluorescence confocal microscopy as described in Materials and Methods. Flag-TAK1 was detected by incubating cells with anti-FLAG M2 and subsequently with Alexa Fluor® 594 goat anti-mouse antibodies. (A) Localization of EGFP-TIP27 transfected alone into COS-1 cells; (B) localization of EGFP-TIP27 co-transfected with TAK1; (C) localization of TAK1 (Alexa Fluor® 594) co-transfected with TIP27; (D) overlap of the EGFP and Alexa Fluor® 594 images; and (E) DIC image.
Figure 8
Figure 8
(A) and (B) TIP27, but not TIP27(ΔTID), functions as a repressor of DR1-dependent transcriptional activation by TAK1 and has little effect on RORγ- and PPARα-mediated transactivation. COS-1 cells were transfected with (DR1)3-CAT, CMV-βGal, TAK1 and increasing amounts of pcDNA4-TIP27 expression vectors as indicated. Another series were transfected with (UAS)5-LUC, pFR-LUC, Gal4(DBD)RORγ(ΔN41) or Gal4(DBD)PPARα(LBD) expression vectors in the presence or absence of pcDNA4-TIP27 (0.1 μg). The PPARα agonist GW327647 (100 nM) was added after 6 h to cells transfected with Gal4(DBD)PPARα(LBD). After 48 h, cells were collected and assayed for reporter activities as described in Materials and Methods. The relative reporter activity was calculated and plotted. (C) TIP27 does not affect VP16 transactivation function and TAK1 homodimerization. CV-1 cells were transfected with (UAS)5-LUC, pM-TAK1 and VP16-TAK1 in the absence and presence of TIP27 expression vector (0.15 μg) as indicated. (D) TIP27 does not inhibit binding of TAK1 to DR1. COS-1 cells were transfected with (DR1)3-CAT and VP16-TAK1 in the absence and presence of TIP27 expression vector (0.15 μg).
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