Proteomic Analysis of Steady-State Nuclear Hormone Receptor Coactivator Complexes
Sung Yun Jung,
Anna Malovannaya,
Jinsong Wei,
Bert W. OMalley and
Jun Qin
Department of Molecular and Cellular Biology (S.Y.J., A.M., J.W., B.W.OM., J.Q.), Verna and Mars McLean Department of Biochemistry and Molecular Biology (S.Y.J., J.W., J.Q.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Jun Qin, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: jqin{at}bcm.tmc.edu.
 |
ABSTRACT
|
---|
We report our initial efforts in the analysis of endogenous nuclear receptor coactivator complexes as a research bridging strand of the Nuclear Receptor Signaling Atlas (NURSA)
(www.nursa.org).
A proteomic approach is used to systematically isolate a variety of coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry. We have isolated and identified seven coactivator complexes including the p160 steroid receptor coactivator family, cAMP response element binding protein-binding protein, p300, coactivator of activating protein-1 and estrogen receptors, and E6 papillomavirus-associated protein. The newly identified coactivator-associated proteins provide unbiased clues and links for understanding of the endogenous hormone receptor coregulator network and its regulation. We hope that the electronic availability of these data to the general scientific community will facilitate generation and testing of new hypotheses to further our understanding of nuclear receptor signaling and coactivator functions.
 |
INTRODUCTION
|
---|
HORMONES AND BIOACTIVE metabolites regulate development and physiology primarily through transcriptional modulation. Nuclear receptors (NRs), which are ligand-dependent and DNA sequence-specific transcription factors, and coactivators that have no specific DNA-binding affinity, are two defining components for the eucaryotic endocrine transcriptional program (1). Receptors recruit coactivators in a ligand-dependent manner to accomplish activation and regulation of transcription. A large number of NR coactivators have been identified by cloning methods based on protein-protein interactions. One group of coactivators, the p160 coactivator family consisting of SRC-1 [steroid receptor coactivator-1; nuclear receptor coactivator (NCoA)-1] (2, 3, 4, 5), SRC-2 [steroid receptor coactivator-2; transcription intermediary factor (TIF)-2; glucocorticoid receptor-interacting protein (GRIP)-1; nuclear receptor coactivator (NCoA)-2] (2, 3, 6) and SRC-3 [steroid receptor coactivator-3; p300/CBP-interacting protein (p/CIP); receptor-associated coactivator (RAC)-3; activator for thyroid hormone and retinoid receptor (ACTR); amplified in breast cancer (AIB)-1; thyroid hormone receptor activator molecule (TRAM)-1] (4, 7, 8), plays an early and direct role in nuclear receptor action. Another group of general coactivators, including acetyltransferases CREB binding protein (CBP), p300 (9, 10), and p300/CBP-associated factor (p/CAF) (11, 12), and methyltransferases such as coactivator-associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1) (13), function in general transcription as well as in hormone action. Coactivators can also regulate cotranscriptional processes such as RNA splicing (14). In addition, similar to well-studied transcription factors, coactivators are the targets of signal transduction pathways that regulate gene sets (12). As a group, coactivators play diverse roles ranging from histone modification, chromatin remodeling, and transcriptional regulation to signal transduction from the environment to the genome. Current estimates reveal that there are approximately 150 different coactivators that have been suggested to play a role in NR actions in cells
(www.nursa.org).
It is clear now that most coactivators reside in large protein complexes and that these coactivator complexes may be considered to be functional units or machines (1, 15). One goal of the Nuclear Receptor Signaling Atlas
(NURSA)
project is to understand this complex hormone receptor coregulator network, its regulation, and the signaling pathways by which these machines communicate. As a first step to dissect the cellular NR coactivator network, we are using a proteomic approach to systematically purify coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry (MS). The existence of steady-state coactivator complexes was previously proposed (16, 17, 18). The identification of components of coactivator complexes will provide the backbone for understanding the steady-state NR coactivator network. As suspected, these analyses have provided novel and unbiased insights into coactivator functions in cells and cellular signaling among coactivator complexes.
 |
RESULTS
|
---|
General Considerations in the Purification and Identification of Coactivator Complexes
The relatively low cellular abundance of coactivators generally dictates the use of more than 109 cells for purification procedures. This limited our choice of cell line to one that is easily and less expensively grown in suspension. Consequently, we decided to use HeLa cells.
HeLa cells are the most frequently employed cells for transfection and assessment of NR function. Several coactivator complexes have been purified from HeLa cells (17, 18, 19). Because most of the coactivators are ubiquitously expressed and are employed by a variety of different transcription factors, we reasoned that coactivator complexes in HeLa cells should be representative of the overall cellular biology of coactivators. The limitation of the HeLa cell is that it is only one cell type, and it does not express several NRs. However, considering the magnitude of the problem (i.e. number of coactivators) and the expense, it is the only logical first approach. If a future coactivator of interest is not expressed in HeLa cells, we will purify it from a cell line that does express the protein.
We decided to use both affinity tagging and antibody affinity methods to purify the coactivator complexes. These two methods compliment each other. Antibody affinity allows the purification of endogenous complexes but often suffers from some antibody cross-reactivity in which an antibody recognizes proteins that share similar epitopes to the antigen used in raising the antibody. It is also limited by the availability of high-affinity antibodies that can immunoprecipitate sufficient amount of protein of interest. Whereas affinity tagging solves the cross-reactivity problem, it is limited by the ability to establish a stable cell line with the tagged protein. It becomes difficult to establish a stable cell line if the expressed protein is detrimental to the growth of the cell, as coactivators frequently are. Another technical limitation is that the most commonly used retrovirus packaging limits the size of the gene to 3 kb. When the gene is larger, the efficiency of retrovirus packaging decreases dramatically, rendering the process of stable cell line generation inefficient. In our first approach to purify endogenous complexes, we raised two to four peptide antibodies to screen for antibodies that effectively immunoprecipitate the antigen. If we fail to find multiple high-affinity antibodies, we will establish stable cell lines for proteins smaller than 100 kDa; for proteins larger than 100 kDa, we will make glutathione-S-transferase fusion proteins to generate protein antibodies and screen for high-affinity antibodies.
Although the majority of coactivators function in the nucleus, some of them are localized to the cytoplasm, where they may receive regulation signals from signal transduction pathways (20, 21). Because the study of proteomics dictates an unbiased approach, we decided to purify coactivator complexes from both nucleus and cytosol.
We chose 14 coactivators for this initial study to test the efficacy of antibody affinity for the isolation coactivator protein complexes. We made 30 peptide antibodies, carried out more than 100 immunoprecipitations (IPs) and analyzed more than 1000 bands with MS. Screening of these antibodies resulted in 14 antibodies (
50% success rate) against seven coactivators that allow isolation of coactivator immunocomplexes from cytosolic extract (S100) and/or nuclear extract (NE). Schematics of these coactivators with known domain structures and some known interacting proteins are illustrated in Fig. 1
. The locations of the peptides that were used to raise antibodies are also depicted (Fig. 1
). Our analyses may define new roles for coactivators in substeps of transcription and in cellular signaling.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1. The Location of Peptides Used for Antibody Generation
Antibodies were generated by immunizing rabbits with short peptides located in the indicated exons. Domain structure of the coactivators and some of known interacting proteins are shown.
|
|
The Issues of Antibody Cross-Reactivity and Nonspecific Associated Proteins
Antibodies may cross-react with other proteins that contain similar epitopes and need to be distinguished from associated proteins. We used two criteria to examine antibody cross-reactivity. First we performed Western blot analysis of cytoplasmic and nuclear fractions to determine whether proteins other than the antigen were evident. As shown in Fig. 2
, a majority of the antibodies recognized a single band corresponding to the expected size of the antigen. This suggests that the antibodies are specific for the protein of interest. We also took a more rigorous approach and examined immune precipitates from cytoplasmic and NEs under denaturing conditions followed by MS to identify co precipitating proteins. We reasoned that under denaturing conditions the antibody should bring down cross-reacting proteins, whereas an associated protein might be disrupted in this environment and not precipitate under these conditions. As shown in Fig. 3
, in addition to the antigen, a small number of proteins are also immunoprecipitated, suggesting that these factors are either cross-reacting or tightly associated proteins. As will be seen in upcoming figures, the number of proteins associated with the cofactors identified under nondenaturing conditions is significantly greater than under denaturing conditions, suggesting that a majority of the proteins identified represent associated factors rather than cross-reacting proteins.

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 2. Characterization of Antibody Specificity with Western Blotting
HeLa NE and S100 fractions were separated on SDS-PAGE and blotted with anti-SRC-1, anti-SRC-2, anti-SRC-3, anti-CBP, anti-p300, anti-E6-AP and anti-CAPER antibodies at 1:5000 dilution. Exposure time varies depending on signal intensity.
|
|

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 3. Characterization of Antibody Specificity with RIPA-IP
The NE and S100 fractions were diluted with 1 vol RIPA and immunoprecipitated with indicated antibodies. Precipitated proteins were separated on SDS-PAGE and identified by MS.
|
|
The cytoskeletal proteins including spectrin, myosin, actin, and annexin (see Table 1
) appear due to protein precipitation during incubation of protein A beads with the antibody. This is most likely an accidental event due to the meta-stable nature of the protein extract. Cytoskeletal precipitate is observed sporadically even when the experiments are carried out using the same extracts, by the same operator, and at the same time.
It is also crucial to distinguish between specific and nonspecific binding proteins. Strictly speaking, until a functional interaction can be demonstrated, the binding should be considered putative. To limit the number of proteins for which functional interactions need to be evaluated, we decided to use an empirical approach to define nonspecific proteins as proteins that repetitively copurify with many unrelated complexes. These nonspecific proteins are listed in Table 1
. In the work reported here, we tentatively assign proteins as coactivator-associated proteins only when they meet the following criteria: 1) the associated bands have weaker intensity than that of the antigen in the large-scale nondenaturing protein extract IP; 2) they are lost in the RIPA-IP; and 3) in general, they are not in Table 1
.
Isolation of p160 SRC Complexes
SRC-1/NCoA-1.
A representative example for the isolation of SRC-1-associated proteins using antibodies is shown in Fig. 4
. Two antibodies, BL434 and BL435, were used to IP SRC-1 from S100 and NE. Because a small amount of SRC-1 was immunoprecipitated from S100, we concluded that copurified proteins from S100 are most likely to be cross-reacting or nonspecific binding proteins. These two antibodies yield similar, but not identical, associated proteins from NE (compare lanes 3 and 4). A common set of proteins was identified in both BL434 and BL435 IP (see Table 2
), but two additional proteins, PACT/RBBP6 and ITCH were present only in BL 435 IP. PACT/RBBP6 contains a Ring finger domain and ITCH contains a HECT (Homology to E6AP Carboxyl Terminus) domain, both of which are likely to function as E3 ubiquitin (Ub)-ligases. In addition to the above two E3 Ub-ligases, NY-REN-45 is present. It is an antigen recognized by autologous antibodies in patients with renal-cell carcinoma, and it contains a BTB (broad complex, tramtrack and bric a brac) domain that has also been recognized recently to function as an E3 Ub-ligase (22). Two other proteins that are present in both IPs and are involved in the Ub pathway are the hyperplastic discs protein homolog and valosin-containing protein (VCP). The hyperplastic discs protein homolog contains one HECT (homology to E6-AP carbonyl terminus)-type E3 ubiquitin-protein ligase domain, and is reported to be induced by progestin in T-47D breast cancer (23). VCP is a family member of putative ATP-binding proteins involved in ubiquitin-dependent protein degradation by 26S proteasome. VCP binds polyubiquitinated proteins and targets them for proteasome degradation (24). These results reveal SRC-1 to be highly associated with enzymes present in the ubiquitination/26S proteasome pathway.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 4. Identification of SRC-1 Immunocomplex
SDS-PAGE separation of SRC-1 immunocomplexes isolated from HeLa NE and S100 fractions using different anti-SRC-1 antibodies. The numbered associated bands correspond to the proteins listed in Table 2 .
|
|
SRC-2/TIF2/GRIP-1/NCoA-2.
Two antibodies recognize SRC-2. BL436 immunoprecipitates SRC-2 in NE, but cross-reacts with proteins in S100 (Fig. 3
). BL437 immunoprecipitates SRC-2 in S100 (Fig. 5
and Table 2
), but cross-reacts with proteins in NE (Fig. 3
). In the case of SRC-2, kinases were identified as prominent interacting proteins. The SRC-2 S100 immunocomplex contains the TBK1 (TANK-Binding Kinase 1) kinase, a protein that is important for the cellular response to inflammation and infectious agents (25, 26); the SRC-2 NE immunocomplex contains a DNA damage response checkpoint kinase, ATR (Ataxia Telangiectasia and Rad3-related protein) (27).

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 5. Identification of SRC-2 and SRC-3 Immunocomplexes
SDS-PAGE separation of SRC-2 and SRC-3 immunocomplexes isolated from HeLa NE and S100 fractions using different anti-SRC-2 and -3 antibodies. The numbered associated bands correspond to the proteins listed in Table 2 .
|
|
SRC-3/p/CIP/RAC3/ACTR/AIB-1/TRAM-1.
Only a few proteins copurify with SRC-3 using BL438 and BL439 antibodies (Fig. 5
and Table 2
). In contrast to SRC-1, a good amount of SRC-3 could be immunoprecipitated from S100. This is consistent with our prior report that SRC-3 is a cytoplasmic coactivator that shifts to the nucleus upon activation (11). SRC-3 seems to associate with the
-subunit (PSME3) of the proteasome activator PA28 complex (28). Our prior MS analyses revealed IKK
, IKKß, and SSA to be associated with SRC-3 (Table 2
). Again, different antibodies pull down different associated proteins. The transcription factor NFAT4 is only observed in the BL439 IP.
Isolation of General Coactivator CBP/p300 Protein Complexes
Screening of antibodies identified that BL440 and BL443 are excellent for immunoprecipitation of CBP and p300 from NE. They are specific to CBP and p300 because no p300 peptides were observed from the IP of CBP, and vice versa. Because CBP and p300 function as coactivators for a diverse group of transcription factors and overexpressed CBP and p300 functionally interact with numerous general transcription factors, NRs, and coactivator proteins as well as basal transcriptional machinery, we expected transcription-related proteins to be their binding partners. To our surprise, many DNA damage response and DNA repair proteins copurify with CBP and p300 (Fig. 6
). Double-strand break repair proteins Mre11/Rad50/NBS1, mismatch repair proteins Msh2/Msh6, and DNA damage response proteins 53BP1 and MDC1 (Mediator of DNA damage Checkpoint 1) are also found in the p300 immunocomplex. Although MDC1 was identified in p300 RIPA-IP, its intensity was significantly diminished. We think that MDC1 may be better categorized as a strong interacting protein rather than a cross-reacting protein. A definitive assignment needs further experiment (see Discussion). Msh2/Msh6, 53BP1, and MDC1 are identified in the CBP immunocomplex as minor components. Zinc finger and BTB domain containing 2 (ZBTB2), and a Kruppel-like zinc finger protein ZNF639 are more prominent than DNA damage response proteins in the CBP immunocomplex. The BTB domain in ZBTB2 suggests that ZBTB2 may function as an E3 Ub ligase. ZNF639 contains a nine C2H2-type zinc finger motif, which is not likely to work as DNA sequence-specific transcription factor (29).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 6. Identification of p300, CBP, and E6-AP Immunocomplexes
SDS-PAGE separation of p300, CBP and E6-AP immunocomplexes isolated from HeLa NE and S100 fractions using different anti-p300, anti-CBP and anti-E6-AP antibodies. *, Degraded CBP. The numbered associated bands correspond to the proteins listed in Table 2 .
|
|
E6 Papillomavirus-Associated Protein (E6-AP)
E6-AP is best known for its role as an E3 Ub-ligase for the degradation of p53 in papillomavirus infected human cells. We previously reported it to be an interacting partner of the liganded PR using the yeast two-hybrid assay; it displays coactivator activity for PR, ER, and other NRs (30). Additionally, inactivation of the ubiquitin ligase activity of E6-AP did not affect its ability to coactivate PR. We found that E6-AP in HeLa cells is mostly localized in cytosol, and the major component of E6-AP IP in S100 is the phosphorylase kinase complex (Fig. 6
). All four subunits,
2, ß,
, and
, of the phosphorylase kinase complex were identified in the BL446 antibody IP. Only small amount of E6-AP was recovered from the HeLa NE, and no associated proteins were identified (data not shown).
Coactivator of Activating Protein-1 and Estrogen Receptors (CAPER) 
CAPER
was originally identified by its interaction with the coactivator activating signal cointegrator-2 (31). CAPER selectively activates AP-1 and ligand bound ER. Interestingly, we found that CAPER coimmunoprecipitated with many splicing factors in NE (Fig. 7
and Table 2
), including the CrkRS kinase, suggesting that CAPER and/or its associated proteins may be subject to regulation by this kinase. The S100 CAPER immunocomplex contains different copurified proteins than those present in the NE (Fig. 7
and Table 2
). In a separate large study, we examined the role of CAPER in RNA splicing in detail. Indeed, we defined both CAPER
and CAPERß as dual coactivators and RNA splicing regulatory proteins (14). Two proteins that contain a Ring finger domain and are homologous to each other, IRF-2BP2A and C14orf4, were also identified. IRF-2BP2A has been reported to be a repressor for IRF-2 dependent transcription (32). The existence of ring finger domains in these proteins points to another potential connection of a coactivator with the Ub pathway. DNA end joining proteins Ku86/Ku70 also reside in the S100 CAPER complex; DNA-PKcs, which normally associates with Ku86/Ku70 tightly in the nucleus, is notably absent.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 7. Identification of CAPER Immunocomplex
SDS-PAGE separation of CAPER immunocomplexes isolated from HeLa NE and S100 fractions using anti-CAPER antibody. The numbered associated bands correspond to the proteins listed in Table 2 .
|
|
SRC-2 and TBK1 Reside in a Biochemical Protein Complex and Synergistically Activate Nuclear Factor (NF)-
B-Dependent Transcription
To substantiate the putative interaction between SRC-2 and TBK1, we examined cofractionation of these two proteins (Fig. 8
). HeLa NE was fractionated through diethylaminoethyl (DEAE) and Mono-Q columns. Fractions containing both SRC-2 and TBK1 after two columns were further separated on a Superose 6 gel-filtration column. SRC-2 and TBK1 coelute with a size of approximately 670 kDa (Fig. 8A
), and SRC-2 can be reciprocally coimmunoprecipitated with TBK1 from fractions 16 and 17 of Superose 6 (Fig. 8B
). Together, our data suggest that SRC-2 and TBK1 reside in a high-molecular-weight protein complex. Because TBK1 plays a role in intracellular inflammatory signaling (26), our results implicate SRC-2 as a coactivator for a part of this response.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 8. SRC-2 and TBK1 Form a Biochemical Protein Complex and Synergistically Activate NF- B-Dependent Transcription
A, HeLa NE was fractionated on diethylaminoethyl (DEAE), Mono-Q and Superose 6 columns. The elution profiles of SRC-2 and TBK1 were monitored by Western blotting. LS, Loading sample; PT, pass-through. B, Coimmunoprecipitation of SRC-2 with TBK1 in the reciprocal TBK1-IP. C, 293T cells were transfected with either SRC-2 or TBK1 expression vector alone or in combination. Where indicated, the transfected cells were treated with recombinant human TNF- (20 ng/ml) 16 h after transfection for 4 h. Whole cell lysates were assayed for luciferase activity.
|
|
To assess the functional relevance of the SRC-2 and TBK1 interaction, we tested whether SRC-2 and TBK1 can synergistically activate NF-
B activity using a reporter gene containing three copies of the NF-
B-responsive element (igk-luc). Transfection of SRC-2 or TBK1 alone resulted in a weak increase in the luciferase activity. In contrast, cotransfection of SRC-2 with TBK1 resulted in marked synergistic activation in the absence of TNF-
(Fig. 8C
). In the presence of TNF-
, the overall reporter activity was higher but synergistic activation activity between TBK1 and SRC-2 was diminished. We interpreted that TBK1 overexpression has a similar effect to that of TNF-
treatment, suggesting that TNF-
-activated TBK1 enhances the coactivator activity of SRC-2.
 |
DISCUSSION
|
---|
We tested the feasibility of using an integrated proteomic approach that combines antibody affinity purification with MS to isolate and identify NR coactivator complexes and reconstruct the nuclear hormone coactivator signaling network. Using this approach, we isolated and identified seven NR coactivator complexes (SRC-1, SRC-2, SRC-3, p300, CBP, E6-AP, and CAPER) from 14 chosen targets.
There is no prior report in the literature describing the isolation of these seven coactivator complexes. Our experiments identified more than 60 proteins that we deem specifically associated with these seven coactivators. It is striking that proteins in the Ub pathway are greatly prominent in the list of these coactivator-associated proteins. More than 10 proteins containing motifs for an E3 Ub-ligase, Ub binding, and protein degradation are found as coactivator-associated proteins. Among the p160 SRC family coactivators, the diversity of associated proteins suggests that functional specifications occur and that these different coactivator complexes play distinct roles in transcriptional regulation. It is intriguing that proteins in the Ub pathway are overrepresented among the putative SRC-1- and SRC-3-interacting proteins. We postulate that one of the major functions of SRC-1 and SRC-3 is to bring the ubiquitination machinery to promoters via interaction with NRs in the course of transcriptional activation. Our past studies with E6-AP (E3) and UBCH7 (E2) and NRs are consistent with this physiologic role (33). The identification of a selective association of SRC-2 with a host defense kinase TBK1 suggests that SRC-2 may function as a transcription coactivator in host defense. Previously, we have shown that IKK
and IKKß specifically associated with SRC-3 and were critical for activating SRC-3 and for ER in breast cells (20, 21). Together, these studies suggest that p160 coactivators are subject to regulation via phosphorylation and bring a variety of enzymatic activities (e.g. acetyltransferases, ubiquitylation enzymes, etc.) to promoters when they are recruited by NRs and other transcription factors.
It was surprising that CBP and p300 associate with multiple proteins in DNA repair and DNA damage response pathways. This observation may reflect an important role of CBP and p300 in maintaining genome integrity by providing requisite HAT activities in the context of the chromatin environment. It is tantalizing to speculate that these DNA repair and DNA damage response factors also play active roles in transcriptional activation. Thus, CBP/p300 may provide a common need for both DNA repair and transcriptional activation by modifying chromatin structure and its environment.
The seven coactivator complexes we analyzed in this study are quite different from other more general coactivator complexes such as the p/CAF and SMCC complexes (15, 34), which we have analyzed previously. The coactivator complexes described herein are not very stable and their associated proteins are readily disrupted. We feel that associated proteins in these NR coactivator complexes represent weaker and more transient interactions, in contrast to the p/CAF and SMCC coactivator complexes that demonstrate relatively stable interactions. Such transient interactions should allow greater flexibility for combinatorial regulation of transcription. It is likely that the p160 coactivators and CBP/p300 transiently interact with many transcriptional regulatory proteins in the cell to provide more generally required enzymatic activities. In our experiments, we are likely to have isolated the relatively more stable subpopulation of the weaker interacting partners that are able to survive our biochemical manipulations. We suggest that in the organization of the cellular coactivator network, the p160 and CBP/p300 coactivators may belong to the category of dating hubs (14), whereas the SMCC/mediator type coactivators may represent the more stable party hubs (14).
It is generally accepted that antibodies raised against different regions of the same protein are capable of immunoprecipitating different proteins. This can occur for at least two reasonsdifferent antibodies recognize different epitopes of the protein and may either select the protein complex conformation in which the epitope is exposed, or the antibody itself may displace binding proteins. It is quite possible that different antibodies against the same antigen can purify different protein complexes. Consequently, the more different types of antibodies are used, the more likely it is to isolate different heterogeneous protein complexes. At present, we do not have enough epitope tagging data to compare its efficacy with antibody affinity data. Our efforts to epitope tag SRC-1 and SRC-3 to overexpress and purify these complexes have been unsuccessful because stable overexpression of SRC/p160 family members is toxic to the cell. The use of the tet-on system allowed us to isolate stable cell lines and immunoprecipitate SRC1 and SRC-3, but few associated proteins could be purified, probably because the expression levels of SRC1 and SRC3 were too high for their binding partners to form biochemically stable and isolatable complexes.
One of our goals was to establish a ground rule for differentiating real associations from cross-reacting and nonspecific binding proteins. This still remains a challenging task. As mentioned before, we attempted to use both Western blotting and IP in RIPA buffer to avoid misidentifying cross-reacting proteins as associated proteins. It appears that IP in RIPA buffer is a better method for this purpose. However, it creates another complicationcross-reacting proteins cannot be distinguished from stable associations that survive in the presence of high concentration of detergent. For example, the double-strand DNA repair complex Rad50-Mre11-NBS1 complex cannot be disrupted by the RIPA buffer (our unpublished observation). Thus, if equal amount of protein (e.g. DDX24 in the p300 primary IP) is present in both nondenaturing IP and RIPA-IP, it could be either a cross-reacting protein or stably associated protein. We envision that it is necessary to carry out reciprocal IPs (e.g. DDX24) to obtain a definitive answer. If the primary protein is recovered in the reciprocal complex, we can conclude that it is a stably associated protein. This will increase the workload, but is necessary for constructing a coactivator-interacting network.
In comparison to cross-reacting proteins, it is relatively easy to distinguish nonspecific binding proteins. We found a group of proteins that often coprecipitate in many seemingly unrelated protein complexes. As indicated in Table 1
, they may not appear all the time in every IP, but different subsets of them are always present. We tentatively assign them as nonspecific proteins. One should consider the possibility that genuine associated proteins may be dismissed as nonspecific proteins. For example, heterogeneous ribonuclear proteins often appear in different IPs and were generally considered as nonspecific proteins. However, when splicing factor complexes are purified, these criteria should be applied with caution. Therefore, Table 1
should only be used as a guide and evaluated on an individual basis.
Validation of new coactivator-associated proteins discovered with this proteomic approach is critical for general application. Toward this end, we have analyzed a considerable number of associated proteins and have found functional connections with parent coactivators for every associated protein (seven of seven) identified by the mass spectrometric analyses that we examined in detail. One of the first surprises was the association of IKK
and IKKß specifically with SRC-3. We recently published that these kinases, previously unknown to play any role in NR or coactivator function, were critical for activating SRC-3 and for ER activity in breast cells (20, 21). Interestingly, the antibodies used in this SRC-3 coimmunoprecipitation provided some additional associated proteins compared with the SRC-3 antibody employed in Fig. 5
; this, again, highlights the relative antibody selectivity discussed in General Considerations. In another example, we analyzed the steady-state coactivator complex associated with the CAPER
. We found numerous splicing factors, indicating that this coactivator plays a role in RNA splicing. Indeed, we validated this observation and published that CAPER
and CAPER ß are dual-function coactivators, capable of both enhancing transcription and regulating target gene alternative RNA splicing (35). In the current study, we found that TBK kinase immunoprecipitated with SRC-2 and validated its association and involvement in coactivation by SRC-2 (see Fig. 8
). We also found that TBK1 phosphorylates SRC-2 in response to infectious agents, and that SRC-2 is required for activation of endogenous viral response genes (our unpublished data).
Although it is physically impossible to analyze all of the proteins associated with the coactivator complexes we isolated, we currently have accumulated many additional examples demonstrating the efficacy of this approach that are in various stages of analyses in the lab. Some unpublished examples are as follows: we validated SSA, a ring finger protein found to be associated with SRCs, to play a role in turnover of the SRC coactivators; PSME3 (REG
) associates with SRC-3, and we have shown that it plays a direct role in linking SRC-3 with the proteasome; MDC-1 is a functional coactivator-associated protein that may link steroid receptors with the DNA repair apparatus. In each of the above cases, we have verified the coactivator binding and the transcriptional function of the associated protein. In summary, we have been successful in validating seven of seven interactions uncovered by our proteomic analyses. This is extraordinarily high considering that it is always difficult to design the appropriate test for proteins that have no previously known functions in hormone action.
We predict that proteomic approaches will continue to uncover a diverse and unexpected biology for coactivators and their associated proteins. Importantly, the NURSA
project will now provide these results to an open electronic forum
(www.NURSA.org)
where investigators worldwide can use such information. Our proteomics effort in dissecting the signaling network of NR coactivators is an integral component of a grand undertaking aimed at understanding the systems biology of NRs. The initial endeavor presented here has convinced us that an integrated proteomic approach is capable of identifying the backbone of the entire NR coactivator network. Such information should provide many crucial clues to the scientific community that will allow the generation of new hypotheses and experiments toward a more complete understanding of the diverse biology and signaling pathways of an investigators favorite NR coactivator. With our current high success rate for purifying and identifying coactivator-associated proteins, we now feel that we are in a position to further scale up our operation. Our immediate goal is to define the signaling network for approximately 50 coactivator complexes in the next 18 months and to provide a software package to categorize and understand cellular interactions and signaling pathways among a wide variety of coactivator complexes. In accord with the NIH NURSA
mandate, our entire data set will be made available electronically to the general scientific community in a timely fashion
(www.NURSA.org).
 |
MATERIALS AND METHODS
|
---|
Antibodies
All rabbit polyclonal antibodies were generated at the Bethyl Laboratory (Montgomery, TX) by immunizing rabbits with the corresponding purified synthetic peptides as antigens. The antibodies were affinity purified.
NE and Cytoplasmic Fraction Preparation
NE and cytoplasmic fractions (S100) were prepared by Dignams method (36). One hundred liters of HeLa cell culture were harvested and washed three times with cold PBS. The packed-cell volume (PCV) was measured, and the cell pellet was gently resuspended with 5 PCVs of hypotonic buffer [10 mM HEPES-KOH (pH 8.0), 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)]. The cells were incubated on ice for 10 min and then pelleted by centrifugation at 1800 x g for 10 min. Hypotonic buffer was added to 2 PCVs, and cells were resuspended and then homogenized with 15 strokes using a pestle B in a Dounce glass homogenizer (Wheaton, Millville, NJ) until the cells were more than 90% lysed, as determined by a light microscope. The lysate was centrifuged at 20,000 x g for 30 min at 4 C. The supernatant was saved for cytoplasmic (S100) fraction, and the pellet was saved to measure the packed nuclear volume. Then 0.4 ml of extraction buffer [20 mM HEPES-KOH (pH 8.0), 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (vol/vol) glycerol, 1 mM DTT, 0.2 mM PMSF] per milliliter of packed nuclear volume was added. Cell nuclei were homogenized with 10 strokes of pestle A in a Dounce glass homogenizer. The suspension was stirred at 4 C for 30 min and centrifuged for 30 min at 20,000 x g. The supernatant (NE) was dialyzed against a dialysis buffer [20 mM HEPES-KOH (pH 8.0), 100 mM KCl, 0.2 mM EDTA, 20% (vol/vol) glycerol, 0.2 mM PMSF, 1 mM DTT] for 2 h. The sample was then centrifuged at 20,000 x g for 30 min, and the supernatant (NE) was aliquoted, frozen in liquid nitrogen, and stored at 80 C. For the S100 fraction, the resulting supernatant was mixed with 0.11 volume of high-salt buffer [20 mM HEPES-KOH (pH 8.0), 20% glycerol, 1.2 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF] and centrifuged at 100,000 x g for 1 h at 4 C. The supernatant was dialyzed against dialysis buffer for 2 h at 4 C. The sample was centrifuged at 20,000 x g for 30 min, and the supernatant (cytoplasmic fraction, S100) was aliquoted, frozen in liquid nitrogen, and stored at 80 C. Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA).
Western Blotting
Five and 25 µg of each HeLa NE and S100 were separated on 6 or 8% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The immunoblots were probed with the following antibodies: anti-SRC-1 (BL434, BL435), anti-SRC-2 (BL436, BL437), anti-SRC-3 (BL438, BL439), anti-CBP (BL440), anti-p300 (BL443), anti-E6-AP(BL446) and anti-CAPER (BL462). Horseradish peroxidase-conjugated antirabbit IgG (Bethyl Laboratory) was used as secondary antibody at 1:5000 dilution, followed by chemiluminescence detection.
Immunoprecipitation
The immunoprecipitations were carried out with both NE and S100 extracts.
For antibody characterizations, thawed NE and S100 fractions were diluted with 1 volume of RIPA buffer [150 mM NaCl, 1.0% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM Tris (pH 8.0)] and cleared by spinning at 100,000 x g for 20 min at 4 C. One milliliter of supernatant (
10 mg total protein) was mixed with 20 µg of affinity-purified antibodies and rotated overnight at 4 C. The sample and antibody mixture was then centrifuged at 100,000 x g for 20 min at 4 C. The cleared supernatant was mixed with 50 µl of protein A-Sepharose beads (50% slurry) and rotated for 2 h at 4 C. The immunoprecipitates were washed three times with the 10% RIPA in PBS. The washed beads were boiled with 40 µl of Laemmli buffer and subjected to SDS-PAGE (420% Tris/glycine gel; Invitrogen, Carlsbad, CA).
For complex purification, the NE and S100 were thawed on ice and cleared by spinning at 20,000 x g for 30 min at 4 C. One and a half milliliter of supernatant (
15 mg total protein) was mixed with 20 µg of affinity-purified antibody and rotated for 4 h at 4 C. The sample and antibody mixture was then centrifuged at 15,000 x g for 20 min at 4 C. The cleared supernatant was mixed with 50 µl of protein A-Sepharose beads (50% slurry), and rotated for 1 h at 4 C. The immunoprecipitates were washed three times with the NETN buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40]. The washed beads were boiled with 40 µl of Laemmli buffer and subjected to SDS-PAGE (420% Tris/glycine gel). If an insufficient amount of protein is purified for protein identification from 15 mg of extracts, the same procedure is carried out using 50100 mg of extracts to increase the amount of purified protein.
In-Gel Digestion
The Coomassie brilliant blue-stained protein bands were excised from the gel and subjected to in-gel digestion. Excised gel pieces were destained with 50 mM ammonium bicarbonate solution in 50% methanol. All gel pieces were then washed with 50% methanol and 10% acetic acid overnight and then washed in HPLC water for 4 h. After the wash procedure, gel pieces were digested with 300 ng of trypsin in 50 mM NH4HCO3 (pH 8.5) for 4 h in a volume of 10 µl. After digestion, peptides were extracted by the addition of 30 µl of acetonitrile. The supernatant was removed and the gel pieces were rehydrated with 10 µl of 50 mM NH4HCO3, followed by a second extraction of peptide with 30 µl of acetonitrile. The pooled supernatants were dried in a Speed-Vac dryer (Thermo Savant, Holbrook, NY).
LC/ESI/MS/MS
An electrospray ion trap mass spectrometer (LCQ; Thermo Finnigan, San Jose, CA) coupled online with a capillary HPLC (Magic 2002; Michrom BioResources, Auburn, CA) was used to acquire MS/MS spectra. An 0.1- x 50-mm MAGIC C18 column (5 µm, 200 A pore size) with mobile phases of A (methanol: water:acetic acid, 5:95:1) and B (methanol:water:acetic acid, 85:15:1) was used with a gradient of 298% of mobile phase B over 2.5 min followed by 98% B for 2 min at a flow rate of 150 µl/min. The flow was split with a Magic precolumn capillary splitter assembly (Michrom BioResources) and 1 µl/min directed to the 100-µm column. The dried peptide sample was dissolved in 8 µl of mobile phase A and 2 µl was injected in HPLC. The ion trap mass spectrometer was operated in a data-dependent fashion during the liquid chromatography run, in which the mass spectrometer switch to MS/MS mode to acquire MS/MS spectra once the intensity of a peptide ion exceeded a preset value. The MS/MS spectra were used to identify proteins by the database search program PepFrag (http://prowl.rockefeller.edu/). All MS/MS spectra were verified manually.
Transfection and Luciferase Assay
293T cells were seeded 24 h before transfection at 3 x 105 per well in a 24-well dish. The cells were transiently transfected with a total of 0.4 µg of empty vector or the indicated plasmids (a combination of 0.1 µg of NF-
B-luciferase and 0.3 µg of indicated expression vector or empty vector as filler DNA) using Lipofectamine 2000 as specified by the manufacturer (Invitrogen). Where indicated, the transfected cells were treated with recombinant human TNF-
(20 ng/ml; Roche Molecular Biochemicals, Indianapolis, IN) 16 h after transfection for 4 h. Whole cell lysates were assayed for luciferase activity using the Dual-Luciferase reporter assay system as instructed by the manufacturer (Promega, Madison, WI). The Renilla-luciferase reporter gene (50 ng) was used as an internal control.
 |
ACKNOWLEDGMENTS
|
---|
We thank members of the Qin and OMalley labs for discussion and technical assistance. We thank National Cell Culture Center for providing cells.
 |
FOOTNOTES
|
---|
This work was supported by the Nuclear Receptor Signaling Atlas (DK62434) and HD08818 grants from the National Institutes of Health.
First Published Online July 28, 2005
Abbreviations: CAPER, Coactivator of activating protein-1 and estrogen receptors; CBP, cAMP response element binding protein (CREB)-binding protein; DTT, dithiothreitol; E6-AP, E6 papillomavirus-associated protein; IPs, immunoprecipitations; MDC1, mediator of DNA damage checkpoint; MS, mass spectrometry; NCoA, nuclear receptor coactivator; NE, nuclear extract; NF, nuclear factor; NR, nuclear receptor; NURSA, Nuclear Receptor Signaling Atlas; p/CAF, p300/CBP-associated factor; PCV, packed-cell volume; PMSF, phenylmethylsulfonyl fluoride; SRC, steroid receptor coactivator; Ub, ubiquitin; VCP, valosin-containing protein; ZBTB2, BTB domain containing 2.
Received for publication November 23, 2004.
Accepted for publication May 20, 2005.
 |
REFERENCES
|
---|
- McKenna NJ, OMalley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465474[CrossRef][Medline]
- Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol. 17:27352744
- Voegel JJ, Heine MJ, Tini M, Vivat V, Chambon P, Gronemeyer H 1998 The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J. 17:507519
- Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
- Onate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:13541357[Abstract]
- Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA. 93:49484952
- Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569580[CrossRef][Medline]
- Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965968[Abstract/Free Full Text]
- Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signalling. Nature 383:99103[CrossRef][Medline]
- Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403414[CrossRef][Medline]
- Blanco JC, Minucci S, Lu J, Yang XJ, Walker KK, Chen H, Evans RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a nuclear receptor coactivator. Genes Dev. 12:16381651
- Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
- Koh SS, Chen D, Lee YH, Stallcup MR 2001 Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem. 276:10891098
- Han JD, Bertin N, Hao T, Goldberg DS, Berriz GF, Zhang LV, Dupuy D, Walhout AJ, Cusick ME, Roth FP, Vidal M 2004 Evidence for dynamically organized modularity in the yeast protein-protein interaction network. Nature 430:8893[CrossRef][Medline]
- Gu W, Malik S, Ito M, Yuan CX, Fondell JD, Zhang X, Martinez E, Qin J, Roeder RG 1999 A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol Cell 3:97108[CrossRef][Medline]
- Ito M, Roeder RG 2001 The TRAP/SMCC/mediator complex and thyroid hormone receptor function. Trends Endocrinol Metab. 12:127134
- McKenna NJ, Nawaz Z, Tsai SY, Tsai MJ, OMalley BW 1998 Distinct steady-state nuclear receptor coregulator complexes exist in vivo. Proc Natl Acad Sci USA. 95:1169711702
- Goo YH, Sohn YC, Kim DH, Kim SW, Kang MJ, Jung DJ, Kwak E, Barlev NA, Berger SL, Chow VT, Roeder RG, Azorsa DO, Meltzer PS, Suh PG, Song EJ, Lee KJ, Lee YC, Lee JW 2003 Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins. Mol Cell Biol. 23:140149
- Fondell JD, Ge H, Roeder RG 1996 Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc Natl Acad Sci USA. 93:83298333
- Wu RC, Qin J, Hashimoto Y, Wong J, Xu J, Tsai SY, Tsai MJ, OMalley BW 2002 Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) coactivator activity by I
B kinase. Mol Cell Biol. 22:35493561
- Wu RC, Qin J, Yi P, Wong J, Tsai SY, Tsai MJ, OMalley BW 2004 Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways. Mol Cell 15:937949[CrossRef][Medline]
- Xu L, Wei Y, Reboul J, Vaglio P, Shin TH, Vidal M, Elledge SJ, Harper JW 2003 BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425:316321[CrossRef][Medline]
- Callaghan MJ, Russell AJ, Woollatt E, Sutherland GR, Sutherland RL, Watts CK 1998 Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs. Oncogene 17:34793491[CrossRef][Medline]
- Dai RM, Li CC 2001 Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat Cell Biol. 3:740744
- McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Maniatis T 2004 IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci USA. 101:233238
- Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, Coyle AJ, Liao SM, Maniatis T 2003 IKK
and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 4:491496
- Abraham RT 2001 Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15:21772196
- Murata S, Kawahara H, Tohma S, Yamamoto K, Kasahara M, Nabeshima Y, Tanaka K, Chiba T 1999 Growth retardation in mice lacking the proteasome activator PA28
. J Biol Chem. 274:3821138215
- Imoto I, Yuki Y, Sonoda I, Ito T, Shimada Y, Imamura M, Inazawa J 2003 Identification of ZASC1 encoding a Kruppel-like zinc finger protein as a novel target for 3q26 amplification in esophageal squamous cell carcinomas. Cancer Res. 63:56915696
- Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, OMalley BW 1999 The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol. 19:11821189
- Jung DJ, Na SY, Na DS, Lee JW 2002 Molecular cloning and characterization of CAPER, a novel coactivator of activating protein-1 and estrogen receptors. J Biol Chem. 277:12291234
- Childs KS, Goodbourn S 2003 Identification of novel co-repressor molecules for interferon regulatory factor-2. Nucleic Acids Res. 31:30163026
- Verma S, Ismail A, Gao X, Fu G, Li X, OMalley BW, Nawaz Z 2004 The ubiquitin-conjugating enzyme UBCH7 acts as a coactivator for steroid hormone receptors. Mol Cell Biol. 24:87168726
- Ogryzko VV, Kotani T, Zhang X, Schiltz RL, Howard T, Yang XJ, Howard BH, Qin J, Nakatani Y 1998 Histone-like TAFs within the PCAF histone acetylase complex. Cell 94:3544[CrossRef][Medline]
- Dowhan DH, Hong EP, Auboeuf D, Dennis AP, Wilson MM, Berget SM, OMalley BW 2005 Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPER
and CAPERß. Mol Cell 17:429439[CrossRef][Medline]
- Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:14751489