Novel transcription coactivator complex containing activating signal cointegrator 1

Abstract

Human activating signal cointegrator 1 (hASC-1) was originally isolated as a transcriptional coactivator of nuclear receptors. Here we report that ASC-1 exists as a steady-state complex associated with three polypeptides, P200, P100, and P50, in HeLa nuclei; stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor kappaB (NF-kappaB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the previously described transrepression between nuclear receptors and either AP-1 or NF-kappaB. Interestingly, ectopic expression of Caenorhabditis elegans ASC-1 (ceASC-1), an ASC-1 homologue that binds P200 and P100, like hASC-1, while weakly interacting only with p65, in HeLa cells appears to replace endogenous hASC-1 from the hASC-1 complex and exerts potent dominant-negative effects on AP-1, NF-kappaB, and SRF transactivation. In addition, neutralization of endogenous P50 by single-cell microinjection of a P50 antibody inhibits AP-1 transactivation; the inhibition is relieved by coexpression of wild-type P50, but not of P50DeltaKH, a mutant form that does not interact with P200. Overall, these results suggest that the endogenous hASC-1 complex appears to play an essential role in AP-1, SRF, and NF-kappaB transactivation and to mediate the transrepression between nuclear receptors and either AP-1 or NF-kappaB in vivo.

FIG. 1.

Interaction of ASC-1 with SRF, p50, p65, and c-Jun. (A) Indicated proteins were labeled with [³⁵S]methionine by in vitro translation and incubated with glutathione beads containing GST alone (−) or fusions of GST to c-Jun, c-Fos, p50, p65, SRFΔC, SRFΔN, SRF, and ASC-1. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. (B) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described previously (2). (C) HeLa cells were transfected with a lacZ expression vector, a Gal4-LUC reporter construct, and an increasing amount of VP16-ASC-1 expression vector (in nanograms).

FIG. 2.

ASC-1 as a coactivator of AP-1, NF-κB, and SRF (A) and as a mediator of transrepression between nuclear receptor and either AP-1 or NF-κB (B). HeLa cells were transfected with the lacZ expression vector and increasing amounts of the ASC-1 expression vector along with the indicated reporter genes. For activation, the expression vector for p65 or c-Jun or serum shock (i.e., addition of 20% fetal bovine serum to cells deprived of serum) was applied, as indicated (in nanograms). Solid and open bars (B), absence and presence, respectively of 100 nM 9-cis-retinoic acid (RA). RXR, retinoic X receptor.

FIG. 3.

Purification of the ASC-1 complex in vivo. (A) HiTrap Q column fractionations reveal distinct elution profiles for different factors as indicated. RXR, retinoic X receptor. (B) Overall purification schemes for the ASC-1 complex summarized. Ab, antibody. (C) An immunoaffinity-purified ASC-1 complex from HeLa cells was resolved in SDS-polyacrylamide gel and stained with Coomassie blue. The sizes of marker proteins (M) are in kilodaltons.

FIG. 4.

Confirmations for the authenticity of the isolated cDNAs. (A) Antibodies were generated against polypeptides encoded by cDNAs which were isolated based on the peptide sequencing data of the purified proteins. + and −, HiTrap Q fractions containing the ASC-1 complex from HeLa nuclear extracts and unrelated fractions, respectively. Each antibody recognized a protein with the expected molecular weight (MW; in thousands). (B) The ASC-1 complex fraction (HiTrap Q) was immunoprecipitated with anti-P50 or anti-ASC-1 antibody. Equivalent amounts of load (Ld), supernatant (S), and pellet (P) from the immunoprecipitation (IP) were blotted and probed with antibodies to the proteins indicated at the right. Asterisks, protein bands nonspecifically immunoreactive with the anti-P100 antibody. (C) HiTrap Q fractions were subjected to Western blotting (WB) using the antibodies indicated. P200, P100, and P50 were cofractionated in this and other column fractionations (data not shown). FT, flowthrough.

FIG. 5.

(A, C, and D) Amino acid sequences of P200, P100, and P50. The peptide sequences originally obtained from mass spectrometry are underlined. The italicized boldface residues within P100 and P50 represent residues deleted in P100s and a KH domain (24), respectively. (B) Duplicated RNA helicase domains in P200. The RNA helicase consensus sequences are as described previously (29). h, hydrophobic residue; Nt, N terminus; Ct, C terminus.

FIG. 6.

Interactions among the ASC-1 complex components. (A) Northern blot analyses indicate that P200, P100, and P50 are ubiquitously expressed as previously described for ASC-1 (11). s. muscle, smooth muscle; s. intestine, small intestine; P.B.L, peripheral blood lymphocytes. (B, C) Plasmids encoding the indicated B42 and LexA proteins were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described previously (2). The Western analysis with the anti-P100 antibody showed comparable levels of expression of P100 and P100s in yeast (data not shown). β-Gal., β-galactosidase. (D) ASC-1 was cotranslated in vitro with either P100 or P100s in the presence of [³⁵S]methionine and immunoprecipitated with an anti-P100 or anti-ASC-1 antibody. (E and F) HeLa cells were transfected with the lacZ expression vector and increasing amounts of P100, P100s, P50, and P50ΔKH expression vector along with indicated reporter genes. For activation, expression vectors for p65 and c-Fos were also cotransfected, as indicated (in nanograms).

FIG. 7.

P200-ASC-1 interaction interfaces and essentiality of P50 in AP-1 transactivation. (A) A combination of fusions of B42-ASC-1 and LexA to wild-type P200 or P200 deletion mutants (left panel) and fusions of B42 to the wild-type ASC-1 or ASC-1 deletion mutants and LexA-P200 (right panel) were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described previously (2). None of the LexA-P200 constructs exhibited autonomous transactivation function (data not shown). +++, strongly blue colonies after 2 days of incubation; ++, light blue colonies after 2 days of incubation; +, light blue colonies after more than 2 days of incubation; −, white colonies. Gray bars, deduced interaction domains. Two copies of DExH-type helicase domains, the helicase superfamily C-terminal domains (HELIc), and SEC63 domains of unknown function within P200 as well as the zinc finger (ZNF) domain of ASC-1 are indicated. (B) (Left) Specificity of the P50 antibody was demonstrated by specific immunoprecipitation (IP) of P50 from a mixture of in vitro-translated and radiolabeled constituents of the ASC-1 complex as well as specific detection of P50 from HeLa nuclear extract in Western analysis (WB). Ld and pre, 20% of the reaction mixture and preserum, respectively. (Middle) Photographs of FITC-stained injected cells (top) and the corresponding pattern of X-Gal (5-bromo-4-chloro-3-indolyl-β-d-thiogalactopyranoside) staining (bottom) with microinjection of either control IgG or anti-P50 IgG. The presence or absence of 0.1 μM TPA is indicated. (Right) The number of cells that express lacZ relative to the total number of FITC-positive cells microinjected with either control IgG or anti-P50 IgG. The presence or absence of TPA (0.1 μM) and a P50, P50ΔKH, or Gal4-VP16 expression vector is indicated. The reporter constructs were AP-1-lacZ and Gal4-lacZ. Experiments were repeated twice with similar results (error range, 5 to 10%), with >200 cells injected.

FIG. 8.

Interaction profiles of ceASC-1. (A) Schematic representations of hASC-1, mASC-1, and ceASC-1 as well as two chimeric ASC-1 constructs. The putative zinc finger domain and the amino acid numbers for each construct are indicated. The relative percentages of similarity to hASC-1 for the central zinc finger domain and the N- and C-terminal domains are shown. (B) In the GST pull-down assays, radiolabeled ceASC-1 was incubated with GST alone or fusions of GST to p65, p50, c-Fos, c-Jun, and SRF. The fusion of LexA to ceASC-1 was cotransformed into yeast cells along with B42 alone or B42 fusion proteins, as indicated. β-Gal., β-galactosidase. (C) Transiently expressed ceASC-1 was copurified with other subunits of the hASC-1 complex. The HiTrap Q column fraction containing HA-tagged ceASC-1 (see Materials and Methods) was resolved in a Superose 6 sizing column, and the fractions were analyzed by immunoblotting with antibodies to the proteins indicated at the right (anti-HA for ceASC-1). (D) Coimmunoprecipitation of ceASC-1 with other ASC-1 complex subunits, but not with hASC-1. The HA-ceASC-1-containing column fraction (HiTrap Q) was immunoprecipitated with an anti-100, anti-ASC-1, or anti-HA antibody. Twenty percent of the load (Ld) and supernatant (sup) and 100% of the pellet from the immunoprecipitation (IP) were blotted and probed with antibodies to the proteins indicated at the right.

FIG. 9.

Dominant-negative phenotype of ceASC-1. (A and B) HeLa cells were transfected with the lacZ expression vector and increasing amounts of ceASC-1, hASC-1, chASC-1, and hcASC-1 expression vectors along with the indicated reporter genes. For activation, expression vectors for p65 and c-Fos were also cotransfected, as indicated. +, serum shock; h, ce, ch, and hc, hASC-1, ceASC-1, chASC-1, and hcASC-1, respectively. (C) Schematic representation of the ASC-1 complex. P200 binds both ASC-1 and P50, and ASC-1 also binds P100. The N-terminal region of P200 has not been isolated yet; it may make additional contacts with other components. Cotransfected ceASC-1 in HeLa cells appears to replace the endogenous ASC-1 from the complex (Fig. 8C and D) and acts as a dominant-negative mutant, likely due to the inability of ceASC-1 to properly interact with target transcription factors (see the text for further discussion).