Modulation of glucocorticoid receptor-interacting protein (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domains Pei-Yao Liu1, Tsai-Yuan Hsieh2, Wei-Yuan Chou1 and Shih-Ming Huang1 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan Department of Medicine, Division of Gastroenterology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Keywords co-activation; GRIP1; HDAC1; nuclear receptor; transactivation Correspondence S.-M Huang, National Defense Medical Center, Department of Biochemistry, 161, Section 6, MinChuan East Road, Taipei, Taiwan 114 Fax: +886 287924057 Tel: +886 227937318 E-mail: shihming@ndmctsgh.edu.tw (Received 11 December 2005, revised 13 March 2006, accepted 16 March 2006) doi:10.1111/j.1742-4658.2006.05231.x Glucocorticoid receptor-interacting protein (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region AD1 activity appears to be mediated by CBP ⁄ p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase (CARM1) The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1 Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions Members of the nuclear receptor (NR) superfamily are ligand-inducible transcription factors This family includes the receptors for steroids, thyroid hormone and vitamin D, as well as orphan receptors for which no ligands have yet been identified [1,2] Each receptor has two activation functions (AFs), namely hormone independent (AF-1) and hormone dependent (AF-2) The relative importance of AF-1 and AF-2 varies between different NRs and is influenced by ligand, cell type and the target gene promoter [3,4] The mechanism by which DNA-bound NRs regulate transcription appears to involve the recruitment of co-regulatory proteins, including co-activators and co-repressors [5–8] Co-activators are not usually DNA-binding proteins, but are recruited to the promoter through protein–protein contact with transcriptional activators Transcriptional co-repression can involve competition for limiting factors, displacement of positive factors, or histone deacetylation to generate a chromatin structure that limits promoter accessibility [7,8] Therefore, the latest working model regarding transcriptional regulation by NRs is an initial association with transcriptional Abbreviations ACTR, activator for thyroid hormone and retinoid receptors; AD, activation domain; AF, activation function; AR, androgen receptor; CARM1, co-activator-associated arginine methyltransferase 1; CoCoA, coiled-coil co-activator; ER, estrogen receptor; GAC63, GRIP1-associated co-activator 63; GAL4DBD, Gal4 DNA-binding domain; GRIP1, glucocorticoid receptor-interacting protein 1; GST, glutathione S-transferase; HA, hemagglutinin; HAT, histone acetyltransferase activity; HDAC1, histone deacetylase 1; HMT, histone methyltransferase; NR, nuclear receptor; RLU, relative light unit; SRC-1, steroid receptor co-activator 1; TR, thyroid receptor; TSA, trichostatin A 2172 FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-Y Liu et al co-repressors, followed by recruitment of co-activators in response to ligands and other signals [9] There are at least three families of NR co-activators: CBP ⁄ p300; the p160 family; and p ⁄ CAF [7,10,11] The best characterized of these is a family of three structurally related, but genetically distinct, 160 kDa proteins called the NR co-activators or p160 co-activators [12–18] These three proteins are steroid receptor co-activator (SRC-1), glucocorticoid receptor-interacting protein (GRIP1, also called TIF2), and activator for thyroid hormone and retinoid receptors (ACTR) (also called RAC3, pCIP, AIB1 and TRAM1) These co-activators bind directly to the DNA-bound NRs and apparently function by recruiting secondary co-activators, such as CBP ⁄ p300, co-activator-associated arginine methyltransferase (CARM1), or related proteins, and possibly by acetylating or methylating histones or other proteins involved in the transcription machinery [5,10,19–21] Two separate domains of p160 co-activators can bind to AF-1 and AF-2 of NRs The p160 co-activators contain at least three NR-interacting boxes or LXXLL motifs (where L stands for leucine and X can be any amino acid) in their central regions, which interact directly with the highly conserved AF-2 domain of NRs [22] The C-terminal region of the p160 co-activator can interact with the AF-1 domain of some NRs and enhance their AF-1 activities in the absence of ligands [23–25] Recent studies have identified three activation domains (which transduce the activation signal) in the p160 co-activator [23,26,27] The enhancement of NR activity by the p160 co-activator depends on the CBP ⁄ p300 family, which is necessary for the function of activation domain (AD1) (amino acids 1075–1083 in GRIP1) [26] AD1 receives an activating signal from DNA-bound NRs and recruits CBP ⁄ p300 [23,27] CBP ⁄ p300 may activate the transcription machinery through its histone acetyltransferase (HAT) activity, which acetylates histones and other proteins involved in transcription [28] The second activation domain of the p160 co-activator, AD2, is located in its far C-terminal region (amino acids 1305–1462 in GRIP1) [23,26] The mechanism of signalling by AD2 may involve the weak HAT activity found in two p160 family members (SRC-1 and ACTR), but not in GRIP1 [14,20] The importance of HAT activity for p160 co-activator function has not been established, and no efficiently acetylated substrates have yet been reported CARM1 is a protein with histone methyltransferase (HMT) activity It mainly binds to the C-terminal region of GRIP1 to stimulate its AD2 transactivation function [19] Furthermore, CBP and CARM1 also Autoregulation of GRIP1 functions via C-terminal region support synergistic cross-talk through their HAT and HMT specificities for histones and other transcriptional factors [19,21] A third activation domain, AD3, was recently identified in the highly conserved N-terminal bHLH-PAS domain of p160 co-activators by recruitment of secondary co-activators, including coiled-coil co-activator (CoCoA) and GRIP1-associated co-activator 63 (GAC63) [29,30] As CoCoA and GAC63 have no obvious sequence homology, the nature of their downstream targets and the specific components of the transcriptional machinery remain unknown The mechanisms by which the p160 co-activators function in NR transcriptional activation, and how they are regulated, are not fully understood, and their components have not been identified in detail It remains to be established whether the functions of the p160 co-activator are modulated by post-translational events, such as self-association, protein modification, or subcellular localization In this article, we present several lines of evidence that demonstrate the functional roles of the GRIP1 C terminus in the regulation of its own transactivation and of NR co-activator activities, which are mediated through its repression and self-association properties Hence, our results provide insights into the regulatory mechanisms controlling the functional activities of GRIP1 They extend our understanding of the importance of the structural status of GRIP1 in modulating NR functions Results Autoregulation of GRIP1 AD activities by its C-terminal region Previous studies have demonstrated that deletion of the AD1 or AD2 domain of GRIP1 results in selective loss of its co-activator functions in the NR system, affecting specific primary or secondary co-activator functions [23,26] We were interested in establishing whether this involved the structural components of GRIP1 Therefore, we created various GRIP1 fragments fused with the yeast Gal4 DNA-binding domain (Gal4DBD) and monitored Gal4-responsive reporter (GK1 reporter) luciferase activity in HeLa cells to assess the transactivation activity of the fragments (Fig 1A,B) In general, the reporter activity of fulllength GRIP1 (amino acids 5–1462) was negatively regulated by its structural component (Fig 1, histogram 2, compare A and B) We performed western blotting analysis to examine the expression levels of Gal4 fusions and GRIP1 fragments and found poor expression of full-length GRIP1 (Fig 1C, lane 2), FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS 2173 Autoregulation of GRIP1 functions via C-terminal region P.-Y Liu et al Luciferase Activity A (RLU 10 ) 1462 AD2 AD1 1121 AD1 1462 AD2 AD1 563 13x 563 563 277x 1121 AD1 15x 858x 1304 16x 1462 AD2 2x 1305 1462 AD2 10x AD1 1122 Luciferase Activity B (RLU 10 ) 120 [Gal4DBD; pM vector] 563 1121 AD1 1013 AD1 Mr 1462 AD2 10 160 1x 1013 1121 AD1 C 40 1x [Gal4DBD; pM vector] 35 858x 29000x Mr 10 55 40 100 72 33 55 24 WB anti-Gal4DBD 10 WB anti-HuR Fig Modulation of glucocorticoid receptor-interacting protein (GRIP1) transactivation activity (A, B) Expression vectors (0.5 lg) for the indicated fragments of GRIP1 fused to the Gal4 DNA-binding domain (Gal4DBD) were transiently transfected into HeLa cells together with the GK1 reporter gene (0.5 lg), which encodes luciferase and is controlled by the Gal4 response element The luciferase activity of transfected cell extracts was determined Numbers beside the bars indicate fold activation compared with that of the Gal4DBD alone RLU, relative light units These data are the average of three experiments (mean ± SD; n ¼ 3) (C) COS-1 cells were co-transfected with various Gal4DBD.GRIP1 fragments (2 lg) in a six-well plate Cell lysates were subjected to western blotting analysis and then immunoblotted with anti-Gal4DBD (upper panel) to determine the GRIP1 expression level and anti-HuR (bottom panel) to determine the loading control Results shown are representative of three independent experiments 2174 HDAC1 is involved in the GRIP1 repression complex 290x 72 170 130 which is also evident from Figs 2B and 5C Although the expression of GRIP1 fragments varied, their transactivation activities were primarily determined by structural components For example, a C-terminal truncated GRIP1 (amino acids 5–1121) showed greater reporter activity than one N-terminal truncated GRIP1 (GRIP1-563–1462) (Fig 1A, compare histogram with histogram 4), suggesting a repression region in its C terminus Subsequently, the region encompassing amino acids 1122–1304 was identified as the major repression region in the GRIP1 C terminus (Fig 1A, compare histogram with histogram 6) Furthermore, GRIP1-1013–1121 induced maximal AD1 activity (Fig 1B, histogram 9, compare A and B), which suggests that amino acids 563–1012 also constitute a repression region for AD1 activity (compare histogram with histogram of Fig 1B) Similar patterns of transactivation activity were exhibited by these GRIP1 fragments in human embryonic kidney 293 cells, and the identities of AD1, AD2, and at least two repression regions in amino acids 563–1012 and 1122–1304, were consistent with our findings derived from HeLa cells (data not shown) Having established the existence of a repression property of GRIP1, we investigated whether deacetylase activity mediated through the histone deacetylase (HDAC) family was involved in the repression effect First, we treated HeLa cells with 100 ngỈmL)1 trichostatin A (TSA), an inhibitor of HDAC activity [31], and monitored the changes in reporter activity of Gal4DBD fused with various GRIP1 fragments after 16 h of TSA treatment (Fig 2A) TSA enhanced the reporter activity of the GRIP1 C-terminal fragment (amino acids 1122–1462) (4.5-fold) and suppressed that of full-length GRIP1 (Fig 2A) We then used glutathione S-transferase (GST) pull-down analysis to examine which of the co-repressor proteins, HDAC1, HDAC4, mSin3a or SMRT-a, were involved in the repression complex We found that HDAC1, mSin3a and SMRTa interacted physically with two C-terminal fragments (amino acids 1122–1462 or 1305–1462) (data not shown) In addition, we examined the GRIP1–HDAC1 complex using a co-immunoprecipitation assay in COS7 cells We detected the GRIP1–HDAC1 complex by immunoprecipitating GRIP1 using a hemagglutinin (HA) antibody or by immunoprecipitating HDAC1 using a myc antibody (Fig 2B) HA antibody immunoprecipitation identified three HDAC1-interacting regions, in GRIP1 residues 563–1121, 5–765, and FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-Y Liu et al Fig GRIP1 physically and functionally interacts with histone deacetylase (HDAC1) (A) Expression vectors (0.5 lg) for the indicated fragments of GRIP1 fused to the Gal4 DNA-binding domain (Gal4DBD) (pM vector) were transiently transfected into HeLa cells along with the GK1 reporter gene (0.4 lg) in the absence or presence of 100 ngỈmL)1 trichostatin A (TSA) for 16 h Numbers above the bars indicate fold activation compared with that of no TSA treatment (B) COS-7 cells were co-transfected with various Gal4DBD.GRIP1 fragments (5 lg) and with HDAC1.myc (5 lg) in a 100 mm Petri dish Cell lysates were subjected to immunoprecipitation with anti-myc (upper panel) immunoglobulin and then immunoblotted with anti-hemagglutinin (middle panel) and anti-myc (bottom panel) immunoglobulin for the loading control for GRIP1 and HDAC1 proteins Results shown are representative of three independent experiments (C) Expression vectors (0.4 lg) for the indicated fragments of GRIP1 fused to the Gal4DBD were transiently co-transfected into HeLa cells, together with the GK1 reporter gene (0.2 lg) with 0.2 lg of wild-type pcDNA3.HDAC1.flag (open column) or the enzyme-dead HDAC1 mutant (grey column) The luciferase activity of the transfected cell extracts was determined These data (A,C) are the average of three experiments (mean ± SD; n ¼ 3) Autoregulation of GRIP1 functions via C-terminal region A [Gal4DBD; pM vector] 2x 1462 AD2 AD1 0.3x 1462 AD2 1122 4.5x IP by α-HA B HA + HA.GRIP1 5-1462 + HA.GRIP1 5-1121 + HA.GRIP1 563-1121 + HA.GRIP1 563-1462 + HA.GRIP1 1122-1462 + HA.GRIP1 5-765 + HDAC1.myc + + + + + ++ HDAC1.myc 1122–1462 (Fig 2B, compare lanes 1, 4, 6, and 7) The myc immunoprecipitation also contained these GRIP1 fragments (data not shown) Our results with TSA (Fig 2A) suggested that the HDAC family might be involved in repression through a deacetylase-independent pathway Hence, we used a mutant HDAC1 protein that lacks deacetylase activity and found that the partially repressive effect on the Gal4 reporter activity was the same as with wild-type HDAC1 for both full-length GRIP1 (amino acids 5–1462) and C-terminal GRIP1 (amino acids 1122–1462) in HeLa cells (Fig 2C, compare the histograms with open and grey columns) WB by α-myc α-myc kDa Input (5%) 220 97.6 66 46 30 WB by α-HA HDAC1.myc WB by α-myc Homo-oligomerization of GRIP1 (RLU 10 ) Luciferase Activity C We examined whether the GRIP1 C terminus can interact inter- or intramolecularly with full-length GRIP1 to modulate its transactivation response to other GRIP C-terminal interacting proteins, such as CARM1, Zac1 and ACTN2 [19,32,33] A co-immunoprecipitation assay in COS-7 cells showed that Gal4DBD fused to the full-length GRIP1 (amino acids 5–1462) complexed strongly with HA.GRIP1-563–1462 and weakly with HA.GRIP1-5–765 or HA.GRIP1-563–1121 (Fig 3A, lanes 7, and 8, respectively) Our co-immunoprecipitation analysis suggested that the primary region of GRIP1 self-association is located at its C terminus, within amino acids 1122–1462 (Fig 3A, compare lanes 5–8) In a parallel experiment, we were unable to detect any HA-tag signal by immunoprecipitation using a mouse anti-IgG antibody (Fig 3A, lanes 9–12) Based on the results in Fig 3A, we used GST pull-down none HDAC1 wt HDAC1 mt Gal4DBD Gal4DBD GRIP1 Gal4DBD GRIP1 5-1462 1122-1462 assays to confirm this potential self-association motif with different C-terminal regions of GRIP1 (amino acids 1122–1462, 1305–1462, 1122–1304, 1305–1398, 1305–1462 and 1399–1462) These regions were fused to GST and the fusion proteins were immobilized on agarose beads Their ability to bind to a synthesized FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS 2175 Autoregulation of GRIP1 functions via C-terminal region A IP by α-IgG α -IgG IP by Input 5% α-Gal4DBD α -Gal4DBD HA.GRIP1 5-765 + + HA.GRIP1 5-1 121 HA.GRIP1 563-1462 + HA.GRIP1 563-1 121 + Gal4DBD.GRIP1 5-1462 + + + + P.-Y Liu et al + + + + + + + + + ++ + + + ++ 10 1112 1011 97.6 66 NS 46 WB by α-HA 1305-1462 Input 10% GST 1122-1304 1305-1398 1305-1462 10 1399-1462 1122-1462 GST-GRIP1 GST GST-GRIP1 Input 10% B GRIP1 1122-1462 GST-GRIP11305-1398 GRIP1 GST Input 10% C 1462 765 563 1121 1122 1462 Fig GRIP1 forms a homodimer under in vitro and in vivo conditions (A) COS-7 cells were transfected with the Gal4 DNAbinding domain (Gal4DBD) GRIP15)1462 (5 lg) in the presence of HA.GRIP15)765, HA.GRIP15)1121, HA.GRIP1563)1462, or HA.GRIP1563)1121 (5 lg, in a 100 mm Petri dish) Cell lysates were subjected to immunoprecipitation with anti-Gal4DBD (lanes 5–8) or control (normal mouse IgG) (lanes 9–12) immunoglobulin and then immunoblotted with anti-HA immunoglobulin (B) The protein for the GRIP1 C-terminal region (amino acids 1122–1462) was translated in vitro and incubated with bead-bound glutathione S-transferase (GST)–GRIP1 (amino acids 1122–1462, 1305–1462, 1122–1304, 1305–1398, 1305–1462, and 1399–1462) fusion proteins or with GST alone; bound proteins were eluted, separated by SDS ⁄ PAGE, and visualized by autoradiography (C) The proteins for the GRIP1 fragments were translated in vitro and incubated with bead-bound GST– GRIP11305)1398 fusion protein or GST alone; bound proteins were eluted, separated by SDS ⁄ PAGE, and visualized by autoradiography Results shown are representative of three independent experiments GRIP1 C-terminal fragment (amino acids 1122–1462) was measured in vitro (Fig 3B) The results indicated that amino acids 1305–1398 constitute the primary self2176 association region in the GRIP1 C terminus (Fig 3B, compare lanes 6–10) The amount of protein pulled down by GST–GRIP1-1305–1462 was greater than that pulled down by GST–GRIP1-1122–1462 (Fig 3B, compare lane with 4) GST–GRIP1-1305–1398 was subsequently used to identify whether other GRIP1 regions interact with this C-terminal region in vitro GST–GRIP1-1305–1398 pulled down full-length GRIP1 (amino acids 5–1462) and C-terminal GRIP1 fragments (amino acids 1122–1462) but not N-terminal (amino acids 5–765) or central (amino acids 563–1121) GRIP1 fragments (Fig 3C) Thus, our in vivo and in vitro results suggest that GRIP1 might form at least a homodimer through its C-terminal region Enhancement of GRIP1 AD1 and AD2 activities by an exogenously overexpressed GRIP1 C terminus The recent identification of CARM1, Zac1 and ACTN2 using GRIP1 amino acids 1122–1462 as bait suggests that the GRIP1-dependent co-activation function of these factors might be mediated through a protein–protein interaction with the GRIP1 C terminus [19,32,33] Hence, we examined the effect of exogenously overexpressed full-length GRIP1 (GRIP1-5– 1462), a C-truncated fragment (GRIP1-5–1121) and a C-terminal fragment (GRIP1-1122–1462), on GRIP1 transactivation activity We measured GRIP1 transactivation using the Gal4 reporter activities of full-length GRIP1 and a C-terminal GRIP1 fragment (amino acids 1122–1462) fused with the Gal4DBD vector (Fig 4) The full-length and C-terminal GRIP1 fragments expressed various levels of enhanced reporter activities in the presence of all Gal4DBD-fused GRIP1 fragments (Fig 4A,B) The C-terminal fragment, GRIP1-1122–1462, expressed greater enhancement than full-length GRIP1 only on the Gal4 reporter activity fused with full-length GRIP1, not C-terminal GRIP1 (Fig 4, compare histogram with histogram 4) This suggests that GRIP1-1122–1462 might mediate its enhancement effect on full-length GRIP1 both through its C terminus and through other regions A C-truncated GRIP1 had no or a little enhancement effect on the Gal4 reporter activities (Fig 4, compare histogram and histogram 3) We then used a series of C-terminal truncations to explore the importance of the GRIP1 C-terminal region in the regulation of GRIP1 transactivation activity (Fig 5) The results suggested that residues 1161–1280 constitute the primary repression region for AD1 transactivation activity (Fig 5A, compare histograms 6–9) We also found that GRIP1-truncated fragments associated with full-length GRIP1 in a sequence-dependent FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-Y Liu et al Autoregulation of GRIP1 functions via C-terminal region A B 1122 1462 Gal4DBD AD2 1462 Gal4DBD AD1 AD2 Luciferase Activity (RLU 10 ) 1 1462 AD2 AD1 4 1121 AD1 1122 1462 AD2 10 69x 39x 2.8x 1x 1x [ pSG5.HA vector] Luciferase Activity (RLU 10 ) 111x 1.3x 13x Fig The C-terminal region of GRIP1 is the primary regulatory region for GRIP1 transactivation activities Expression vectors (0.4 lg) for the indicated fragments of GRIP1 (A, amino acids 5–1462; and B, amino acids 1122–1462) fused to the Gal4 DNA-binding domain (Gal4DBD) were transiently transfected into HeLa cells together with the GK1 reporter gene (0.2 lg) in the presence of 0.2 lg of pSG5.HA vector and the indicated fragments of GRIP1 in the pSG5.HA vector The actual luciferase activities measured for each histogram were as follows: for Gal4DBD.GRIP15)1462, 3.3 · 103 ± relative light units (RLU) and for Gal4DBD.GRIP11122)1462, 1.7 · 102 ± 18 RLU Numbers above the bars indicate fold activation compared with that of the ratio related pM.GRIP1 to pM vector These data are the average of three experiments (mean ± SD; n ¼ 3) A B Luciferase Activity (RLU 10 ) 12 15 Fig Residues 1161–1280 are the primary repression region in the GRIP1 C terminus Expression vectors (0.4 lg) for the truncated fragments of GRIP1 fused to the Gal4 DNA-binding domain (Gal4DBD) were transiently transfected into HeLa cells together with the GK1 reporter gene (0.2 lg) (A) in the presence of 0.2 lg of pVP16 vector or pVP16.GRIP1 (B) Luciferase activity of the transfected cell extracts was determined Numbers beside the bars indicate fold activation compared with that of the Gal4DBD vector These data are the average of three experiments (mean ± SD; n ¼ 3) (C) COS-1 cells were co-transfected with various Gal4DBD.GRIP1 fragments (2 lg) in a sixwell plate Cell lysates were subjected to western blotting analysis and then immunoblotted with anti-Gal4DBD (upper panel) for GRIP1 expression and anti-HuR (bottom panel) immunolglobulin for the loading control Results shown are representative of three independent experiments 5 1462 1430 1400 1350 5 1280 1240 1200 1160 1121 10 manner in the mammalian two-hybrid analysis (Fig 5B), and that amino acids 1350–1400 constituted the primary association site of GRIP1 (Fig 5B, com- 1x Gal4DBD pVP16.GRIP1/pVP16 2.7x 3.3x 2.1x 2.6x 10 15 20 25 4x 7 17x 8 28x 46x 10 39x 10 C Gal4DBD.GRIP1 fragment Mr 10 170 130 WB anti-Gal4DBD WB anti-HuR pare histogram with histogram 5) Hence, the enhancement effect on transaction activities of these C-terminal truncations by exogenous full-length or FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS 2177 Autoregulation of GRIP1 functions via C-terminal region P.-Y Liu et al C-terminal GRIP1 also depended on the sequence constitution in the C-terminal region (data not shown) Furthermore, the low expression levels of GRIP1 fragments, such as amino acids 5–1462, 5–1430 and 5–1400 (Fig 5C), suggest that the expression level was not the primary factor because the GRIP1-5–1200 induced higher transactivation activity than GRIP1-5–1350 (Fig 5A,C, compare histograms and lanes with 8) GRIP1 C terminus functions as a GRIP1-dependent NR co-activator in HeLa cells Because the C-terminal region of GRIP1 is involved in the repression of transactivation activity and selfassociation of GRIP1 (Figs 1–5), we examined the relationship between transactivation and co-activation of GRIP1, using a series of C-truncations to monitor its co-activator functions in the androgen receptor (AR), estrogen receptor (ER) and thyroid receptor (TR) systems (Fig 6) Our previous study suggests that GRIP1 AD2 activity is necessary for its co-activation in the AR system, AD1 activity is necessary for its coactivation in the TR system, and cross-talk between AD1and AD2 activities is necessary for maximal coactivation in the ER system [26] We next examined whether the GRIP1 C terminus itself functions as a secondary (or GRIP1-dependent) co-activator, in a manner similar to that of CARM1, in NR transcriptional activation The exogenously co-transfected GRIP1 C terminus, or CARM1 with GRIP1, further enhanced the co-activator function of GRIP1 on various NR transcriptional activations, including AR, ER and TR (Fig 6) In the AR system, the GRIP1 C terminus had a stronger enhancement effect than A B C Fold pSG5.HA 1462 1400 1304 1280 1160 1121 5 765 Fold 10 20 30 40 AR 30 Fold 90 60 ER 40 80 120 160 TR 2 3 4 5 6 7 8 D none GRIP1 1122-1462 CARM1 Mr 170 130 100 72 WB anti-HA WB anti-HuR Fig The GRIP1 C terminus serves as the GRIP-dependent nuclear receptor (NR) co-activator HeLa cells were transfected with the reporter plasmid [0.25 lg of MMTV-LUC vector for androgen receptor (AR) (A), EREII-LUC vector for estrogen receptor (ER) (B), and MMTV[TRE]LUC vector for thyroid receptor (TR) (C)] and the NR expression vector [0.15 lg of AR (A), 0.04 lg of ER vector (B) and 0.04 lg of TR vector (C)] Transfected cells were grown with 100 nM dihydrotestosterone (A), 100 nM estradiol (B) or 100 nM 3,5,5¢-triido-L-thryonine (C) Expression vectors (0.35 lg) for the indicated fragments of GRIP1 fused to the pSG5.HA were transiently transfected into HeLa cells together with GRIP11122)1462 (open column) or CARM1 (grey column) The luciferase activity of transfected cell extracts was determined Numbers beside the bars indicate fold activation compared with that of the pSG5.HA vector alone without co-activator co-transfection These data are the average of three experiments (mean ± SD; n ¼ 3) (D) COS-1 cells were co-transfected with various HA.GRIP1 fragments (2 lg) in a six-well plate Cell lysates were subjected to western blotting analysis and then immunoblotted with anti-HA (upper panel) for GRIP1 expression and anti-HuR (bottom panel) immunoglobulin for the loading control The results shown are representative of three independent experiments 2178 FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-Y Liu et al CARM1 (Fig 6A, compare open with grey columns), whereas CARM1 had a stronger effect on TR transcriptional transactivation than the GRIP1 C terminus (Fig 6C, compare open with grey columns) No GRIP1-dependent TR co-activator effect by the GRIP1 C terminus was observed in GRIP1 fragments containing amino acids 1161–1462 (Fig 6C, compare histograms 2–6, open columns) In the ER transcriptional system, the particular sequence that was truncated determined the effectiveness of the GRIP1 C terminus or CARM1 on GRIP1 co-activator function (Fig 6B, compare open and grey columns) The expression levels of various HA-tag fused GRIP1 fragments were similar to those of the respective Gal4DBD-tag fused GRIP1 fragments, including poor full-length GRIP1 expression (Fig 6D, lane 2) The protein level of GRIP1 fragments was not the primary factor for NR co-activator function, because amino acids 5–765 could not serve as a NR co-activator, even when present at a higher level (Fig 6, compare histograms, and lane with lane 8) Discussion Autoregulation of GRIP1 transactivation activity To date, some of the functions of the N- and C-termini of p160 co-activators were unclear Recently, Stallcup’s laboratory identified two new GRIP1 N-terminal interacting proteins, CoCoA and GAC63 [29,30] In this study, we investigated the regulation of GRIP1 transactivation and co-activation activities by its own C terminus through the repression and selfassociation motifs Our work showed that the major masking effect of the GRIP1 C terminus on GRIP1 transactivation functions could be overcome by exogenous co-expression of the GRIP1 C terminus, but not by the GRIP1 N-terminal fragment (Fig 4) The enhancement of GRIP1 transactivation activities of AD1 and AD2 might be mediated either through truncation or overexpression of its C-terminal region (Figs 1, and 5) These effects differed from those induced by other general GRIP1-dependent co-activators, such as CBP and CARM1 Generally, CBP and CARM1 regulate the co-activator functions of the p160 co-activator in NR systems both through protein–protein interaction and through their catalytic effects (acetylation and methylation, respectively) on histones or other transcriptional factors [19,34–36] There are no reports showing that the C-terminal region of GRIP1 has specific enzymatic activity in modulating basal transcriptional machinery In addition, the effects of CBP or CARM1 on GRIP1 AD1 Autoregulation of GRIP1 functions via C-terminal region or AD2 activity differed from those of the GRIP1 C terminus (data not shown) The repression region of the GRIP1 C terminus might recruit the co-repressor family (Fig 2B and data not shown) The deacetylase inhibitor, TSA, only functioned with the GRIP1 C-terminal fragment (amino acids 1122–1462), and not with full-length GRIP1, suggesting the existence of a mechanism that is different from the deacetylase activity of HDAC1 (Fig 2A) The similarity between the repression effect on GRIP1 transactivation function by wild-type HDAC1 and its enzyme-dead mutant suggested that a protein–protein interaction was involved, not deacetylase activity (Fig 2B,C) GRIP1 associated with its C-terminal region in the co-immunoprecipitation analysis and GST pull-down, but it complexed with the N-terminal and central regions only in the co-immunoprecipitation analysis, not in the GST pull-down analysis (Fig 3) These findings supported the idea that the conformational change of GRIP1 might have resulted from inter- and intramolecular interactions within its C-terminal and other regions Hence, the modulation of GRIP1 transactivation and co-activation activities through its C terminus or other exogenous factors (HDAC1 or CARM1) might be mediated through protein–protein interaction, which change the local conformation of GRIP1 or have downstream effects on basal transcriptional machinery for expressing full GRIP1 co-activator function Figure shows a working model based on our findings The functional roles of the GRIP1 C-terminal region In Figs 1–6, we present several lines of evidence to support the concept that the GRIP1 C-terminal region is involved in the modulation of self-transactivation (AD1 and AD2) and co-activator (AR, ER and TR) functions in HeLa cells The outcome of the relationship between GRIP1 transactivation and co-activator functions varies according to the system under investigation (Figs and 6) In the AR transcriptional system, the GRIP1 co-activator function was destroyed when GRIP1-truncated fragments expressed higher transactivation activity because of the loss of intact AD2-dependent function In contrast, GRIP1 transactivation and coactivation activities were correlated in the TR transcriptional system and the relationship was independent in the ER transcriptional system We also found that the GRIP1 co-activator function depends not only on the existence of a repression domain or a protein–protein interaction with identified and unidentified factors, but also on the GRIP1 conformation under specific FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS 2179 Autoregulation of GRIP1 functions via C-terminal region P.-Y Liu et al 1122 1305 1013 1398 1462 1122 1305 1013 1122 1013 + 1398 1305 1398 1462 1462 14 62 1398 1122 1013 II 1305 1122 1305 I 1398 + 1462 1122 1305 1013 + 1398 1305 1122 1398 1462 1462 1013 1122 1305 1398 1462 + 1462 1398 1122 1122 1013 1305 62 1398 1462 1305 1398 1122 1305 62 1398 1305 III 1122 1013 IV AD1 ? ? Repression domain Association domain 1122 1013 1305 1398 AD2 1462 62 AD3 1122 1398 1305 V Fig Dynamic model of the potential GRIP1 conformational change mediated through its C terminus We propose that either monomeric (I) or dimeric (or higher oligomeric) (II) GRIP1 might form a distinct conformation in cells One repression (grey circle) and association (dotted circle) are defined in this study AD1 (slant circle), (closed circle), and (open circle) have been previously reported [23,26,28] The exposure of any GRIP1 C-terminal interacting protein, including the GRIP1 C terminus in this model, might alter GRIP1 conformation I through intramolecular interaction into conformation III or conformation II through intermolecular interaction into conformation IV (first effect) In this study, the exogenous GRIP1 C terminus dramatically enhanced GRIP1 transactivation activity through the repression and association domains (or the titration of co-repressors), resulting in conformational changes from conformation I (or II) into III or IV In contrast, the extra downstream signal (second effect) of other GRIP1-dependent co-activators might be required for some full GRIP1 NR co-activator functions, for example, the methyltransferase activity of co-activator-associated arginine methyltransferase (CARM1) in this study The question mark indicates that further analyses are necessary to identify the involvement of the GRIP1 N terminus or the status of oligomerization in cells conditions (Figs and 6) Hence, the linking of repression and self-association motifs to the GRIP1 conformation demonstrated in this study might be explained by the effect of the co-expressing GRIP1 C terminus on GRIP1 transactivation and co-activation activities Our western blotting analysis showed that the amount of protein expressed by the exogenous GRIP1 fragment was also tightly regulated by its structural component These findings are consistent with a recent study conducted by the Hager laboratory, which demonstrated that the C terminus of GRIP1 is essential for the formation of discrete nuclear foci and 26S proteasome degradation in gene regulation [37] Similarly to the regulatory mechanism reported in p53 studies [38,39], GRIP1 might form a more active conformation, determined by its relative concentration in cells The relative concentration of GRIP1 might depend on its homo-oligomerization status, which is mainly deter2180 mined by the involvement of its C-terminal region in protein–protein interactions, including self-association, repression by HDAC1 and other proteins, 26S proteasome degradation, or translocalization Taken together, the effect of GRIP1 C-terminal interacting proteins as a GRIP1-dependent secondary co-activator might, in part, be mediated through conformational change of the GRIP1 C terminus and subsequent exposure of a working surface, with extra downstream signalling for its transactivation and NR co-activator functions Experimental procedures Plasmids The pSG5.HA vectors coding for full-length GRIP1 (codons 5–1462), other GRIP1 fragments (codons 5–1121 and 1122–1462), and HA.CARM1 have been described FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-Y Liu et al previously [19]; GRIP1-563–1121 was constructed by inserting an EcoRI–SalI fragment of the appropriate PCR-amplified GRIP1 cDNA into the EcoRI and XhoI sites of the pSG5.HA vector GRIP1-5–765 and GRIP1-1305–1462 were constructed by inserting EcoRI–XhoI fragments encoding GRIP15)765 and GRIP11305)1462 into the pSG5.HA vector; GRIP1-563–1462 was constructed by inserting an XhoI–EcoRI (GRIP1766)1462) fragment from GRIP15)1462 into the pSG5.HA.GRIP1563)1121 treated by XhoI digestion Vectors encoding Gal4DBD fused to various GRIP1 fragments were constructed by inserting EcoRI–SalI fragments of the appropriate PCR-amplified GRIP1 cDNA or EcoRI–XhoI GRIP1 fragments cut from respective pSG5.HA.GRIP1s into the EcoRI and SalI sites of the pM vector (Clontech, Mountain View, CA, USA), a vector for expression of Gal4DBD fusion proteins from a constitutive SV40 early promoter C-terminal truncations of pM.GRIP15)1462 were constructed by inserting XhoI–XbaI fragments of the appropriate truncated PCR-amplified GRIP1 (amino acids from 750 to indicated numbers) into the XhoI and XbaI sites of the pM.GRIP15)1121 vector C-terminal truncations of pSG5.HA.GRIP15)1462 were constructed by inserting EcoRI–SalI fragments of the indicated pM.GRIP1 truncations into the EcoRI and XhoI sites of the pSG5.HA vector Plasmid DNAs encoding pCDNA3.1.HDAC1.myc [40] were gifts from M.A Lazar (University of Pennsylvania, Philadelphia, PA, USA), and pCDNA3.HDAC1.flag wild type and H141A mutant were gifts from T.P Yao (Duke University, Durham, NC, USA) [41] Reporter genes MMTV-LUC, EREII-LUC [GL45], MMTV[TRE]-LUC, and GK1, were as described previously [42,43] The expression of NRs in mammalian cells and ⁄ or in vitro, vectors pSVAR0 for human AR [44], pHE0 for human ERa [43] and pCMX.hTRb1 [9] for human TRb1, were as described previously Bacterial expression vectors for GST fused to various GRIP1 fragments (codons 1122–1462, 1305–1462, 1122– 1304, 1305–1398, 1305–1462 and 1399–1462) were constructed by inserting the appropriate PCR fragment into pGEX-4T1 expression vector (GE HealthCare, Chicago, IL, USA) via EcoRI–XhoI sites Autoregulation of GRIP1 functions via C-terminal region as relative light units (RLU) Luciferase activities are shown as the mean and SD from two transfected sets The results shown are representative of at least three independent experiments Because some co-activators, including GRIP1 and CARM1, enhance the activities of so-called constitutive promoters two- to ninefold, internal controls by co-transfection of constitutive b-galactosidase expression vectors were not used to normalize luciferase data However, internal controls were used strategically to show that variation in transfection efficiency was not a factor in the key results (data not shown) Immunoprecipitation and immunoblots For analysis of the homo-oligomerization of GRIP1 and the physical interaction between GRIP1 and HDAC1, these expression vectors were transfected into COS-7 cells After transfection, cells were lysed in RIPA buffer (100 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl, 0.1% SDS, and 1% Triton 100) at °C Lysates were subjected to immunoprecipitation with antibodies against Gal4 DBD or HA for h, followed by adsorption to Sepharose-coupled protein A ⁄ G (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for h Immunoprecipitates were separated by SDS ⁄ PAGE and analysed with immunoblots For determination of total protein levels of Gal4DBD- or HA-GRIP1 fragments, aliquots of cell lysates were subjected to direct immunoblots Immunoblots were performed as previously described [23] using 10% of the extract from lysates for immunoprecipitation and monoclonal antibodies 3F10 against the HA epitope (Roche, Mannheim, Germany), RK5C1 against Gal4DBD, 3A2 against HuR, and normal mouse IgG (Santa Cruz Biotechnology) Protein–protein interaction assays For GST pull-down assays, 35S-labelled proteins were produced using the TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI, USA) GST fusion proteins were produced in Escherichia coli BL21, eluted, and analysed by gel electrophoresis, as previously described [23] Cell culture and transient transfection assays Acknowledgements HeLa, COS-7 and COS-1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% charcoal ⁄ dextran-treated fetal bovine serum The cells in each well (a six- or a 24-well plate) were transfected with SuperFect Transfection Reagent (Qiagen, Hilden, Germany) or jetPEI (PolyPlus-transfection, Illkirch, France), according to the manufacturer’s protocol; total DNA was adjusted to 2.0 lg (six well) or 1.0 lg (24-well) by addition of the empty vector pSG5.HA Luciferase assays were performed with the Promega Luciferase Assay kit (Madison, WI, USA), and the measurement is expressed numerically We thank Dr W Feng (University of California, USA) for expression vectors and reporter genes for TR; P Webb and P J Kushner (University of California, USA) fro expression vectors and reporter genes for ER; A O Brinkmann (Erasmus University, Rotterdam, the Netherlands) for AR expression vector; M A Lazar (University of Pennsylvania, USA) for pCDNA3.1.HDAC1.myc; and T P Yao (Duke University, USA) for pCDNA3.HDAC1.flag (wild-type and H141A mutant) expression vectors This work FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS 2181 Autoregulation of GRIP1 functions via C-terminal region P.-Y Liu et al was supported by grants from the National Health Research Institute and National Science Council, Taiwan, Republic of China (NHRI-EX94-9224NC and NSC 94-2320-B-016–044 to S M Huang) References Enmark E & Gustafsson JA (1996) Orphan nuclear receptors – the first eight years Mol Endocrinol 10, 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I 13 98 + 14 62 11 22 13 05 10 13 + 13 98 13 05 11 22 13 98 14 62 14 62 10 13 11 22 13 05 13 98 14 62 + 14 62 13 98 11 22 11 22 10 13 13 05 62 13 98 14 62 13 05 13 98 11 22 13 05 62 13 98 13 05 III 11 22 10 13 IV AD1 ? ? Repression. .. FEBS 217 9 Autoregulation of GRIP1 functions via C-terminal region P.-Y Liu et al 11 22 13 05 10 13 13 98 14 62 11 22 13 05 10 13 11 22 10 13 + 13 98 13 05 13 98 14 62 14 62 14 62 13 98 11 22 10 13 II 13 05 11 22 13 05... GST 11 22 -13 04 13 05 -13 98 13 05 -14 62 10 13 99 -14 62 11 22 -14 62 GST-GRIP1 GST GST-GRIP1 Input 10 % B GRIP1 11 22 -14 62 GST-GRIP 113 05 -13 98 GRIP1 GST Input 10 % C 14 62 765 563 11 21 112 2 14 62 Fig GRIP1 forms