Both SRC-1 and SRC-3 exist in protein complexes.
In order to gain more insight into the nature of NR-coactivator complexes, we used biochemical fractionation to ascertain the existence of such complexes. For this purpose, HeLa nuclear extracts were fractionated on a Superose 6 sizing column and the distribution of SRC-1 and SRC-3 in each fraction was analyzed by Western blotting. In agreement with previous results from our laboratory, SRC-1 was eluted in fractions with estimated sizes of 440 to 700 kDa (Fig. ). We found that the majority of SRC-3 was eluted in fractions overlapping with SRC-1 (28
), but the peak concentrations did not coincide (fractions 28 and 30). Additionally, a minor pool of SRC-3 existed in fractions with an estimated size of 1.5 MDa (Fig. , lane 16). Since members of the SRC family of coactivators are about 160 kDa in size, these results indicate that both SRC-1 and SRC-3 proteins exist in large protein complexes.
We next used a combination of conventional gel filtration and antibody affinity chromatography to further purify these SRC-1- and SRC-3-containing complexes (Fig. ). After fractionation of HeLa nuclear extracts on a Q Sepharose column (Pharmacia), both SRC-1 and SRC-3 proteins were found to be present and enriched in the 0.3 M KCl eluate as determined by Western blot analysis (data not shown). This 0.3 M KCl fraction was subjected to further purification by SRC-1 and SRC-3 antibody affinity columns. Following purification on the antibody affinity columns, the resulting SRC-1 and SRC-3 protein complexes were resolved by SDS-10% PAGE and the identities of the associated proteins were determined by MS (42
). As expected, SRC-1 and SRC-3 were eluted from the antibody columns, as confirmed by MS (Fig. ). In addition, the presence of SRC-2 in both SRC-1 and SRC-3 complexes is somewhat interesting, supporting the possibility of heterodimerization of SRC family members, as had been suggested previously (33
). However, the authenticity of this dimerization awaits further verification, as the possible presence of small amounts of antibody cross-reactivity cannot be completely ruled out.
In addition, we also identified CBP and SS-A/Ro autoantigens as common components of both SRC-1 and SRC-3 complexes. CBP had been shown to interact with SRC family members (50
), and the presence of CBP thus supports the validity of the use of antibody affinity purification as a means for identifying associated proteins.
SS-A/Ro contains putative zinc finger domains and a leucine zipper motif at its N-terminal half and has been shown to bind to DNA (9
). Its presence is of specific clinical interest, as antibodies to this protein are common in patients with rheumatic diseases, including systemic lupus erythematosus and Sjogren's syndrome (8
). The function of SS-A/Ro in the complexes is not yet known.
Most interestingly, two components of the IKK complex, IKKβ and IKKγ, were identified exclusively from the SRC-3, but not the SRC-1, protein complex. The IKK complex, composed of the two catalytic subunits IKKα and IKKβ and one regulatory IKKγ subunit (15
), has been shown to phosphorylate IκB in response to proinflammatory stimuli, such as TNF-α, bacterial lipopolysaccharides, or interleukin-1 (15
). Although the third component of the IKK complex, IKKα, was not initially identified by MS from the immunocomplex, a protein corresponding to the molecular mass of IKKα (p85) was visualized by Coomassie blue staining and was subsequently identified as IKKα by Western blot analysis. Taken together, our results identified the complete IKK enzyme as a component of the SRC-3 protein complex.
Association of SRC-3 and IKK.
After determining the identities of these associated proteins, we wished to confirm the capacity of SRC-3 to associate with the identified proteins. Since cofractionation of proteins on a sizing column indicates possible protein association, we fractionated HeLa nuclear extracts on a Superose 6 gel column and analyzed the candidate proteins for coelution with SRC-3 by using Western blotting. As shown in Fig. , CBP, a known SRC-3-interacting protein that was identified here as an SRC-3-associated protein by MS, was found also to cofractionate with the major SRC-3 fractions (fractions 26, 28, and 30) of SRC-3. In addition, the IKKβ and IKKγ subunits of the IKK complex cofractionated with the larger SRC-3 complex, suggesting an association of these proteins in a common complex. However, since the peaks of IKKβ and IKKγ (fraction 24) did not coincide with the main SRC-3 peak, our results indicate that the SRC-3 complex is heterogeneous in nature.
FIG. 2. Confirmation of intracellular association of SRC-3 and IKK subunits. (A) HeLa cell nuclear extracts were fractionated as described in the legend for Fig. . The presence of the indicated proteins was determined by Western blot analysis. (more ...)
To further confirm the association of IKK with SRC-3, we analyzed the presence of IKKβ and IKKγ in the purified SRC-3 immunocomplex by using Western blot analysis. As expected, SRC-1 and SRC-3 were highly and specifically enriched by the immunopurification (Fig. , lane 2 for SRC-3 and lane 5 for SRC-1). More importantly, we found that the SRC-3-specific antibody could coimmunoprecipitate (Co-IP) IKKβ and IKKγ, whereas the SRC-1-specific antibody failed to do so (Fig. , left panel; compare lanes 2 and 5). In support of this finding, the IKKβ-specific antibody not only precipitated IKKβ and IKKα but also SRC-3 in a reciprocal Co-IP experiment (Fig. , right panel, lane 8). As a control, rabbit preimmune serum was used and was found to be unable to precipitate SRC-3, IKKα, or IKKβ under similar conditions (Fig. , lanes 6 and 7).
To further substantiate the association of SRC-3 with the IKK subunits in vivo, we tested their interactions by transiently coexpressing these proteins in HeLa cells. Following coexpression of Flag-tagged SRC-3 and HA-tagged IKKβ in HeLa cells, an immunoprecipitation (IP)-Western blot analysis was performed. As shown in Fig. , cotransfection of Flag-SRC-3 and HA-IKKβ, followed by IP with anti-Flag antibody, resulted in the Co-IP of IKKβ with SRC-3, as demonstrated by Western blot analysis using anti-HA antibody (Fig. , lane 6), whereas IP from control cells or cells which received transfection of either SRC-3 or IKKβ alone failed to do so (Fig. , lanes 1, 2, and 4). We confirmed the presence of IKKα in a similar IP-Western blot experiment. Clearly, both SRC-3 and IKKα were found to Co-IP by Flag antibody when Flag-SRC-3 and HA-IKKα were coexpressed in the same cell but not in cells transfected with either one alone (Fig. , lanes 2, 3, and 5).
To ensure that the association of IKK and SRC-3 interaction is physiological, we examined their association in HeLa cells where the SRC-3-IKK complex was initially identified. For this purpose, we performed a Co-IP with anti-SRC-3 antibody by using both cytoplasmic and nuclear extracts from HeLa cells. We found association of SRC-3 with all three subunits of IKK in both nuclear and cytoplasmic extracts (Fig. , lanes 2 and 4). Taken together, our results indicate that IKK is a genuine component of the SRC-3 complex.
SRC-3 and IKK act in concert to activate NF-κB-mediated transactivation.
We questioned whether NF-κB-mediated gene expression can be synergistically modulated by SRC-3 and IKK. For this purpose, a reporter gene harboring three copies of the NF-κB responsive element was cotransfected with individual expression vectors for SRC-3 and IKK, either alone or in combination. Our results showed that transfection of IKKα, IKKβ, or SRC-3 alone resulted in a three- to sixfold increase in activation of the promoter (Fig. , compare lanes 2, 5, and 9 to lane 1). Importantly, cotransfection of SRC-3 with either IKKα or IKKβ resulted in 17- or 50-fold activation, respectively (Fig. , lanes 6 and 10). These results show that IKK and SRC-3 can synergistically activate NF-κB-dependent transcription.
FIG. 3. Synergistic activation of an NF-κB-responsive promoter by SRC-3 and IKK. HeLa cells were transfected with either SRC-3 or IKK expression vector alone or with the two combined. Where indicated, TNF-α (+) was added 24 h after transfection (more ...)
Since TNF-α activates NF-κB-dependent transcription through modulation of IKK activity, we tested the effects of TNF-α in our system. We found that treatment with TNF-α alone can activate the promoter threefold (Fig. , lanes 1 and 3). In the presence of either SRC-3 or IKK alone, TNF-α was able to moderately enhance promoter activity (Fig. , lanes 2 and 4, 5 and 7, and 9 and 11). In contrast, a much greater enhancement by TNF-α of promoter activity was achieved in the presence of both SRC-3 and IKK (Fig. , compare lanes 7 and 8 and 11 and 12).
IKK kinase activity is required for synergistic activation with SRC-3.
To assess how IKK synergizes with SRC-3 to activate the NF-κB-responsive promoter, we asked whether IKK kinase activity is essential for the activation process. For this purpose, we first tested the ability of an inhibitor of IKK, sodium salicylate (NaSal), to block promoter activation (2
). This inhibitor has been shown to specifically inhibit IKKβ activity but not that of other kinases tested (60
). The results showed that on the addition of NaSal, the activation of the promoter was significantly reduced even in the presence of both SRC-3 and IKK, although it was not completely abolished (Fig. , lanes 6, 8, 14, and 16). As a control, we tested this inhibitor on a promoter that does not contain the NF-κB-responsive element (5× UAS TATA), and the results showed that its activity was not affected (Fig. , left panel).
FIG. 4. Kinase activity of IKK is required for optimum NF-κB activation by SRC-3. (A) HeLa cells were transfected as described in the legend for Fig. . The IKK inhibitor, NaSal, was added to a final concentration of 5 mM after transfection (more ...)
To demonstrate further the importance of kinase activity, we next compared the abilities of the wild-type IKK and a kinase-defective mutant of IKK (62
) to activate the NF-κB promoter. As shown in Fig. , cotransfection of SRC-3 together with the wild-type IKK resulted in greater activation of the promoter than with the mutant IKK counterpart, even in the presence of SRC-3 (Fig. , lanes 9 to 12 and 17 to 20), suggesting that kinase activity is important for activation.
To rule out that the possibility that the defective kinase is simply unable to interact with SRC-3, we tested their interactions in an in vitro pull-down assay. Our results showed that wild-type and mutant kinases interacted equally well with SRC-3 in vitro, suggesting that the inability of the mutants to activate the promoter is due to a decrease in kinase activity and not to an inability to interact with SRC-3 (Fig. , lanes 4, 6, 8, and 10). Taken together, these results strongly suggest that IKK kinase activity is important for SRC-3-enhanced NF-κB gene activation.
Nuclear translocation-stabilization of SRC-3 is induced by TNF-α.
It is known that activation of IKK in response to TNF-α results in nuclear translocation of NF-κB. We showed that TNF-α potentiated SRC-3 activity in NF-κB-mediated gene expression. We therefore investigated the effects of TNF-α on the subcellular distribution of SRC-3. Vehicle-treated cells and cells treated with TNF-α were lysed and fractionated into cytoplasmic and nuclear fractions. These fractions were then subjected to Western blot analysis to determine the distribution of SRC-3. In line with previous reports (54
), our results showed that the majority of SRC-3 was present in the cytoplasmic fractions prior to TNF-α treatment (Fig. , top panel). Interestingly, we found that SRC-3 showed time-dependent translocation to the nucleus from cytoplasm in response to TNF-α. Compared to the vehicle-treated control (2%), up to 33% of SRC-3 was found in the nucleus 1 h after TNF-α treatment (Fig. ). In contrast, TNF-α did not have the same effect on SRC-1, which exists in the nucleus most of the time, suggesting that the translocation is limited to SRC-3 (Fig. , middle panel). As a control for TNF-α action, the degradation of IκB from these cells was monitored. We observed a typical rapid degradation and reappearance pattern (Fig. , bottom panel), which was consistent with earlier reports (16
FIG. 5. Nuclear translocation of SRC-3 is induced by TNF-α. (A) The proteins from untreated cells and cells treated with TNF-α for the times indicated (all grown in DMEM containing 0.5% FBS) were separated into cytoplasmic and nuclear fractions. (more ...) Phosphorylation of SRC-3 by IKK.
Careful analysis of the data showed that the nuclear SRC-3 displayed slower mobility than the cytoplasmic counterpart (Fig. , lanes 1 and 2). The mobility difference was observed again when a mixture of cytoplasmic and nuclear extracts was run side by side with each separated extract (Fig. , lanes 3, 4, and 5). Furthermore, treatment of nuclear extract with λ-phosphatase resulted in faster migration of SRC-3, which is similar to the mobility obtained for cytoplasmic SRC-3 (Fig. , lanes 1, 2, and 3), suggesting that nuclear SRC-3 was phosphorylated. To substantiate this hypothesis, we asked if SRC-3 is a substrate of IKK. For this purpose, we employed an in vitro kinase assay using purified SRC-3 as substrate. Figure shows that SRC-3 was able to be phosphorylated by both IKKα and IKKβ in vitro, with IKKβ phosphorylating SRC-3 more efficiently than IKKα (Fig. , lanes 2 and 3). In addition, we also tested the phosphorylation abilities of the kinase mutants in parallel. Our results showed that the mutants still contained some residual activity and were able to phosphorylate SRC-3 but only at much lower levels (Fig. , lanes 4 and 5). The expected residual kinase activity could explain the slight activation we observed, as shown in Fig. (lanes 10 and 18). The differential phosphorylation of SRC-3 was not due to the inability of mutant IKK to interact with SRC-3 (Fig. ) or to lack of expression, as Western blot analysis showed that similar levels of protein were expressed (Fig. ).
FIG. 6. Phosphorylation of SRC-3 by IKK. (A) HeLa cells were subjected to transfection with the indicated IKK expression vectors, which was followed by immunoprecipitation with anti-HA antibody. The immunoprecipitates were assayed for kinase activity in vitro (more ...)
Since SRC-3 is phosphorylated by IKK in vitro, we next asked whether TNF-α, a physiological inducer of IKK activity, affects the phosphorylation of SRC-3 in vivo. To this end, HeLa cells were labeled with orthophosphate (32Pi) in the presence or absence of TNF-α and then subjected to IP with an anti-SRC-3 antibody to determine whether SRC-3 phosphorylation can be regulated by TNF-α. Our results showed that SRC-3 was a phosphoprotein in vivo and, more importantly, that phosphorylation of SRC-3 was enhanced by TNF-α (Fig. , compare lanes 2 and 4 to 1 and 3). As a control, the levels of SRC-3 expression were left unaltered in the presence or absence of TNF-α (right panel, Coomassie staining). In contrast to SRC-3, our results also showed that phosphorylation of SRC-1 was not affected by TNF-α (Fig. , lanes 5 and 6). Since TNF-α treatment activates IKK, our results support the conclusion that SRC-3 is a target for IKK.
Involvement of SRC-3 in NF-κB-mediated gene expression.
Our data suggested that SRC-3 plays an important role in NF-κB-mediated gene expression (Fig. ) and that its activity is subjected to regulation by phosphorylation. To support the physiological importance of these findings, we investigated the possible role of SRC-3 in immune response. We first examined the expression of the proinflammatory caspases in LNCaP cells, which overexpress SRC-3 in a strictly RU486-inducible manner (7
). The expression of SRC-3 in these cells has been shown to be induced very rapidly by RU486 and reaches maximum levels 2 to 4 h after exposure to the inducing agent (Y. Hashimoto, S. Y. Tsai, and M.-J. Tsai, unpublished result). As shown in Fig. (right panel), induction of SRC-3 by RU486 resulted in the increased expression of two proinflammatory caspases, caspase 4 and caspase 5, as detected by RPA. The induction of caspase expression is rapid and can be detected within the first 4 h upon addition of RU486 and can be maintained for up to 24 h in the continuous presence of RU486. In contrast, no change in the expression of these caspases was detected from the parental LNCaP cells as determined by RPA (left panel).
FIG. 7. NF-κB target gene expression in SRC-3 null animals. (A) Inducible LNCaP cells were grown in the presence (+) or absence (−) of RU486 (100 nM) for the times indicated to induce expression of SRC-3. Afterward, total RNA was prepared (more ...)
Recently, eye and skin infections have been noted in our SRC-3 null mice, suggesting a possible impairment of the immune system in these animals (58
). This result is consistent with a role for SRC-3 in inflammatory responses. To investigate this defect further, we examined the status of IRF-1, a known NF-κB target gene (31
), in SRC-3 null mice. Total RNA prepared from the spleens of control and SRC-3 null mouse littermates was analyzed by RPA to determine whether the levels of IRF-1 mRNA are affected by the genetic deletion of SRC-3. As might be predicted, we found reduced but not abolished expression of IRF-1 in the SRC-3 null mice (Fig. ). In total, we analyzed five mice from each group. Among these, four out of five SRC-3 null mice exhibited significantly reduced basal expression of IRF-1 (Fig. and data not shown). The animal results are also compatible with those for SRC-3 involvement in the regulation of NF-κB-responsive genes in vivo.