Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I kappa B kinase

Abstract

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.

FIG. 1.

Purification of SRC complexes and analysis of associated proteins by MS. (A) HeLa cell nuclear extracts (NE) were fractionated on a Superose 6 sizing column. The presence of SRC-1 and SRC-3 in the indicated fractions was detected by Western blot analysis. Arrows indicate the positions of standard proteins of known molecular weights. Numbers at the top of the panel indicate the fraction number collected. (B) Schematic diagram of protein purification. Ab, antibody. (C) The immunocomplexes resulting from the purification process diagrammed in panel B were resolved by SDS-PAGE and stained with Coomassie blue. Both SRC-1 and SRC-3 complexes are shown. The identities of the indicated proteins from each complex were determined by MS. An asterisk indicates the contaminated heat shock proteins. M, molecular weight markers.

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. 1. The presence of the indicated proteins was determined by Western blot analysis. Numbers at the top of the panel indicate the fraction number collected. Q, Q Sepharose eluate. (B) HeLa cell nuclear extracts were immunoprecipitated with anti-SRC-1 or anti-SRC-3 antibodies. The immunoprecipitates were assayed by Western blot analysis with the indicated antibodies (left panel). For reciprocal coimmunoprecipitation, anti-IKKβ antibody was used in parallel with anti-SRC-3 antibody and the immunoprecipitates were assayed by Western blot analysis with the indicated antibodies (right panel). The input lanes represent 30% of the actual amount for IP. (C) HeLa cells were transfected with the indicated expression vectors. The cell lysates were prepared and subjected to immunoprecipitation with anti-Flag (M2) antibody. The immunoprecipitates were used for Western blot analysis with anti-HA antibody. (D) The cytoplasmic and nuclear extracts from HeLa cells were used for immunoprecipitation with anti-SRC-3 antibody. The presence of the IKK subunits in the immunoprecipitates was confirmed by Western blot analysis with specific antibodies. As a control for immunoprecipitation, the rabbit preimmune serum was used in parallel with anti-SRC-3 antibody and the immunoprecipitates were assayed by Western blot analysis with the indicated antibodies. The input lanes represent 30% of the actual amount for IP.

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. 1. The presence of the indicated proteins was determined by Western blot analysis. Numbers at the top of the panel indicate the fraction number collected. Q, Q Sepharose eluate. (B) HeLa cell nuclear extracts were immunoprecipitated with anti-SRC-1 or anti-SRC-3 antibodies. The immunoprecipitates were assayed by Western blot analysis with the indicated antibodies (left panel). For reciprocal coimmunoprecipitation, anti-IKKβ antibody was used in parallel with anti-SRC-3 antibody and the immunoprecipitates were assayed by Western blot analysis with the indicated antibodies (right panel). The input lanes represent 30% of the actual amount for IP. (C) HeLa cells were transfected with the indicated expression vectors. The cell lysates were prepared and subjected to immunoprecipitation with anti-Flag (M2) antibody. The immunoprecipitates were used for Western blot analysis with anti-HA antibody. (D) The cytoplasmic and nuclear extracts from HeLa cells were used for immunoprecipitation with anti-SRC-3 antibody. The presence of the IKK subunits in the immunoprecipitates was confirmed by Western blot analysis with specific antibodies. As a control for immunoprecipitation, the rabbit preimmune serum was used in parallel with anti-SRC-3 antibody and the immunoprecipitates were assayed by Western blot analysis with the indicated antibodies. The input lanes represent 30% of the actual amount for IP.

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 for 4 h. Thereafter, the luciferase activity derived from the transfected κB-responsive promoter was measured and normalized by total input proteins. The y axis represents relative activities by comparison to those of samples transfected with the NF-κB-responsive promoter alone, whose activity was assigned a value of 1. The bars represent the means and standard deviations of the results from three independent experiments with triplicate samples.

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. 3. The IKK inhibitor, NaSal, was added to a final concentration of 5 mM after transfection and incubated for another 24 h. Thereafter, the luciferase activity was determined and expressed as described earlier. (B) As a control, HeLa cells were transfected with a reporter gene (5× UAS TATA luciferase) along with either GAL4 or GAL4-VP16 expression plasmid, and the results showed that neither the basal (GAL4) (bottom left panel) nor the activated (GAL4-VP16) (bottom right panel) activity of this promoter was affected. RLU, relative light units. SRC-3 and the indicated IKK were cotranslated and labeled with [³⁵S]methionine in vitro. The products were then immunoprecipitated with anti-HA antibody, resolved by SDS-PAGE, and visualized by autoradiography.

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. The membrane containing the fractions was subjected to sequential Western blot analysis by stripping and reprobing the membrane with the indicated antibodies (left panel). The degradation of IκB as evidenced by Western blot analysis was used to monitor the effect of TNF-α on these cells. The results shown are representative of two independent experiments with similar results. (B) Bar chart showing data from experiments as described for panel A. Values corresponding to the y axis represent the percentages of SRC-3 from nuclei (nuclear/nuclear + cytoplasmic [N/N+C]), whereas those corresponding to the x axis represent durations of TNF-α treatment. (C) The proteins from TNF-α-treated HeLa cells were fractionated as described for panel A and assayed by Western blot analysis (left panel). Furthermore, the cytoplasmic fraction was mixed with the nuclear fraction and run side by side with each separated fraction to compare levels of mobility (right panel). (D) Where indicated (+), the nuclear extract was treated with λ-phosphatase (λ-PPase) prior to Western blot analysis. The differences in mobility are indicated by a bracket. N, nucleus; C, cytoplasm.

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 with purified SRC-3 or IκB as a substrate in the presence of [γ-³²P]ATP. The top panel shows the phosphorylated SRC-3 and IκB, and the bottom panel shows the same gel stained with Coomassie blue, indicating the presence of equal amounts of SRC-3 and IκB. Mock, mock transfected. (B) A portion of the immunoprecipitates from the transfections described for panel A was used for Western blot analysis with anti-HA antibody. The results indicated similar amounts of HA-tagged proteins were being precipitated. Mock, mock transfected. (C) HeLa cells were labeled with ³²P_i in vivo in the presence (+) or absence (−) of TNF-α. The lysates were prepared and subjected to immunoprecipitation by anti-SRC-3 antibody. Shown on the left is the extent of SRC-3 phosphorylation, and on the right is the same gel stained with Coomassie blue, which shows that similar amounts of SRC-3 were immunoprecipitated. The experiment was done in duplicate. The phosphorylation of SRC-1 was also determined in a similar manner by using anti-SRC-1 antibody. The presence of TNF-α is indicated by a plus sign. Western blot analysis (WB) was used to determine the SRC-1 protein amount.

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 with purified SRC-3 or IκB as a substrate in the presence of [γ-³²P]ATP. The top panel shows the phosphorylated SRC-3 and IκB, and the bottom panel shows the same gel stained with Coomassie blue, indicating the presence of equal amounts of SRC-3 and IκB. Mock, mock transfected. (B) A portion of the immunoprecipitates from the transfections described for panel A was used for Western blot analysis with anti-HA antibody. The results indicated similar amounts of HA-tagged proteins were being precipitated. Mock, mock transfected. (C) HeLa cells were labeled with ³²P_i in vivo in the presence (+) or absence (−) of TNF-α. The lysates were prepared and subjected to immunoprecipitation by anti-SRC-3 antibody. Shown on the left is the extent of SRC-3 phosphorylation, and on the right is the same gel stained with Coomassie blue, which shows that similar amounts of SRC-3 were immunoprecipitated. The experiment was done in duplicate. The phosphorylation of SRC-1 was also determined in a similar manner by using anti-SRC-1 antibody. The presence of TNF-α is indicated by a plus sign. Western blot analysis (WB) was used to determine the SRC-1 protein amount.

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 with purified SRC-3 or IκB as a substrate in the presence of [γ-³²P]ATP. The top panel shows the phosphorylated SRC-3 and IκB, and the bottom panel shows the same gel stained with Coomassie blue, indicating the presence of equal amounts of SRC-3 and IκB. Mock, mock transfected. (B) A portion of the immunoprecipitates from the transfections described for panel A was used for Western blot analysis with anti-HA antibody. The results indicated similar amounts of HA-tagged proteins were being precipitated. Mock, mock transfected. (C) HeLa cells were labeled with ³²P_i in vivo in the presence (+) or absence (−) of TNF-α. The lysates were prepared and subjected to immunoprecipitation by anti-SRC-3 antibody. Shown on the left is the extent of SRC-3 phosphorylation, and on the right is the same gel stained with Coomassie blue, which shows that similar amounts of SRC-3 were immunoprecipitated. The experiment was done in duplicate. The phosphorylation of SRC-1 was also determined in a similar manner by using anti-SRC-1 antibody. The presence of TNF-α is indicated by a plus sign. Western blot analysis (WB) was used to determine the SRC-1 protein amount.

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 and the expression levels of caspase 4 and caspase 5 were determined by RPA (right panel). The expression of these two caspases from the parental LNCaP cells was also determined by RPA and was shown not to change even in the presence of RU486 (left panel). The levels of L32 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) are shown as a loading control. (B) Total RNA from spleens was analyzed by RPA to determine the expression level of IRF-1. The RPA results for three mice from each group are shown. The level of cyclophilin expression was determined in the same reaction and used as an RNA loading control.

FIG. 8.

Model for SRC-3 regulation by phosphorylation. In response to the presence of TNF-α or ligands, increased phosphorylation of SRC-3 led to the activation and nuclear translocation of SRC-3. The effects of phosphorylation were similar for NF-κB. The association of SRC-3 with IKK was observed in both the cytoplasm and nucleus but is omitted for the nucleus for clarity. The dashed line indicates the putative interaction between SRC-3 and NF-κB (55).