C/EBP beta and Elk-1 synergistically transactivate the c-fos serum response element

Abstract

Background: The serum response element (SRE) in the c-fos promoter is a convergence point for several signaling pathways that regulate induction of the c-fos gene. Many transcription factors regulate the SRE, including serum response factor (SRF), ternary complex factor (TCF), and CCAAT/enhancer binding protein-beta (C/EBPbeta). Independently, the TCFs and C/EBPbeta have been shown to interact with SRF and to respond to Ras-dependent signaling pathways that result in transactivation of the SRE. Due to these common observations, we addressed the possibility that C/EBPbeta and Elk-1 could both be necessary for Ras-stimulated transactivation of the SRE.

Results: In this report, we demonstrate that Elk-1 and C/EBPbeta functionally synergize in transactivation of both a Gal4 reporter plasmid in concert with Gal4-SRF and in transactivation of the SRE. Interestingly, this synergy is only observed upon activation of Ras-dependent signaling pathways. Furthermore, we show that Elk-1 and C/EBPbeta could interact both in an in vitro GST-pulldown assay and in an in vivo co-immunoprecipitation assay. The in vivo interaction between the two proteins is dependent on the presence of activated Ras. We have also shown that the C-terminal domain of C/EBPbeta and the N-terminal domain of Elk-1 are necessary for the proteins to interact.

Conclusions: These data show that C/EBPbeta and Elk-1 synergize in SRF dependent transcription of both a Gal-4 reporter and the SRE. This suggests that SRF, TCF, and C/EBPbeta are all necessary for maximal induction of the c-fos SRE in response to mitogenic signaling by Ras.

Figure 1

Elk-1 and p35-C/EBPβ synergize in SRF dependent transactivation of a GAL4 reporter construct. NIH 3T3 cells were transfected with 1 μg of pG5CAT alone, with 0.5 μg of pGAL4-SRF, or with 0.5 μg of pGAL4-SRF and 0.5 μg of pCMV-LAP (encoding p35-C/EBPβ) and/or 0.5 μg of pCMV-Elk-1 as indicated, in the absence and presence of 1 μg pCMV-Ras.V12. Total DNA in each transfection was adjusted to 3.5 μg with pUC19. Cells were serum deprived for 40 h prior to harvesting. CAT activity was measured as previously described [33]. Data are the average of 14 determinations, and error bars represent standard error.

Figure 2

Elk-1 and p35-C/EBPβ synergize in transactivation of the SRE. NIH 3T3 cells were transfected with 0.75 μg of SRE.CAT and 0.75 μg pCMV-SRF alone or with 0.75 μg pCMV-LAP or 0.75 μg pCMV-Elk1 in the absence or presence of 0.75 μg pCMV-Ras.V12. Cells were serum deprived for 40 h prior to harvesting. CAT activity was measured as previously described [33]. Data are the average of 4 determinations, and error bars represent standard error.

Figure 3

Elk-1 binds to GST-p35-C/EBPβ in vitro. Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p35-C/EBPβ beads (lanes 2) or GST beads (lanes 3) by boiling in Laemmli sample buffer were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-fourth of the amount of the radiolabeled proteins initially mixed with the beads.

Figure 4

Elk-1 coimmunoprecipitates with p35-C/EBPβ, but only in the presence of activated Ras. COS-7 cells were transfected with 10 μg of pCDNA3.1/His-p35-C/EBPβ (lanes 1, 2, 5, 6) and 10 μg of pCMV-Elk-1 (lanes 3, 4, 5, 6) in the presence and absence of 2 μg of pCMV-Ras.V12 as indicated. Cells were harvested 40 h post-transfection, and whole cell lysates were incubated with T7tag Ab-agarose beads, followed by immunoblotting of precipitated proteins with Elk-1 Ab.

Figure 5

The C-terminal domain of C/EBPβ interacts with Elk-1. Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST, GST-p35-C/EBPβ, or GST-p20-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p20-C/EBPβ beads (lane 2), the GST-p35-C/EBPβ beads (lane 3) or GST beads (lanes 4) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.

Figure 6

The A-box of Elk-1 is necessary for interaction with p35-C/EBPβ in vitro. A) Schematic representation of Elk-1 wild-type and deletion mutants. B) Radiolabeled Elk-1(wt), Elk-1(1-209), Elk-1(1-140), and Elk-1(89-428) were prepared by in vitro transcription and translation of their respective expression constructs (see Mat. and Meth.). The proteins were incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST beads (lanes 2) or GST-p35-C/EBPβ beads (lanes 3) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.