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

Abstract

gene. Many transcription factors regulate the SRE, including serum response factor (SRF), ternary complex factor (TCF), and CCAAT/enhancer binding protein-beta (C/EBPβ). Independently, the TCFs and C/EBPβ 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/EBPβ and Elk-1 could both be necessary for Ras-stimulated transactivation of the SRE. interaction between the two proteins is dependent on the presence of activated Ras. We have also shown that the C-terminal domain of C/EBPβ and the N-terminal domain of Elk-1 are necessary for the proteins to interact. SRE in response to mitogenic signaling by Ras.

Background

gene. Many transcription factors regulate the SRE, including serum response factor (SRF), ternary complex factor (TCF), and CCAAT/enhancer binding protein-beta (C/EBPβ). Independently, the TCFs and C/EBPβ 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/EBPβ and Elk-1 could both be necessary for Ras-stimulated transactivation of the SRE.

Results

interaction between the two proteins is dependent on the presence of activated Ras. We have also shown that the C-terminal domain of C/EBPβ and the N-terminal domain of Elk-1 are necessary for the proteins to interact.

Conclusions

SRE in response to mitogenic signaling by Ras.

Introduction

]. This suggests that it is the transcriptional activation of a complex of SRF and its accessory proteins that is regulated rather than regulation of SRF DNA binding.

].

]. Therefore, C/EBPβ is a target of a Ras-dependent signaling pathway that regulates its interaction with SRF.

SRE in response to Ras-dependent signaling pathways.

Elk-1 and p35-C/EBPβ synergize in transactivation of a GAL4 dependent reporter

]. When CMV-Elk-1 is cotransfected with the reporter and GAL4-SRF, there is little increase in CAT activity in the absence of activated Ras (CMV-Ras.V12). This result is expected since the transactivation domain of Elk-1 is activated in response to Ras. Therefore, when activated Ras is transfected with Elk-1, GAL4-SRF, and the reporter, the CAT activity increases to 8-fold over basal levels. We cannot determine if the increase in CAT activity in the presence of Ras also reflects a stimulation of the interaction of the Elk-1 and SRF proteins.

]. Data are the average of 14 determinations, and error bars represent standard error.

]. Thus, the increase in transcription in this assay is due to Ras stimulation of the SRF-p35-C/EBPβ interaction.

Interestingly, when all three constructs - SRF, Elk-1, and p35-C/EBPβ-are cotransfected with the reporter construct, there is an average 260-fold increase in CAT activity in the presence of activated Ras. The values of fold activation varied from as low as 60-fold to as high as 725-fold over basal levels, and we are unsure of the reason for this variability. However, regardless of the extent of activation, in every experiment there was a synergy observed when both Elk-1 and p35-C/EBPβ are transfected in the presence of Ras. There is only a slight increase in CAT activity in the absence of Ras. Therefore, Elk-1 and p35-C/EBPβ are working synergistically to transactivate the reporter construct in the presence of SRF. This synergism is only observed in response to activation of mitogenic signaling pathways by Ras.

Elk-1 and p35-C/EBPβ synergize in transactivation of the SRE

, when p35-C/EBPβ is co-transfected with the reporter construct, there is a 13-fold increase in CAT activity in the absence of activated Ras that is increased to 24-fold when CMV-Ras.V12 is co-transfected.

]. Data are the average of 4 determinations, and error bars represent standard error.

When CMV-Elk-1 is co-transfected with the SRE reporter construct, there is no additional stimulation in transactivation in either the absence or presence of Ras compared to the reporter alone. However, when both Elk-1 and p35-C/EBPβ are transfected with the SRE reporter construct, there is a synergistic effect in transactivation of the SRE, with a 72-fold increase in CAT activity over reporter construct alone. As was seen with the Gal4 reporter, this synergism is only observed in the presence of activated Ras. These data suggest that both Elk-1 and p35-C/EBPβ are necessary for maximal Ras-stimulated transactivation of the SRE.

.

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.

, but only in the presence of activated Ras

, but only in response to activation of Ras-dependent signaling pathways.

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.

.

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.

The N-terminal A-box of Elk-1 is sufficient to interact with C/EBPβ 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.

Discussion

interaction domains of the two proteins are the C-terminal domain of C/EBPβ and the N-terminal domain of Elk-1.

]. However, we show that Ras activation of TCF alone does not result in maximal SRE transactivation, but instead, is greatly enhanced in the presence of p35-C/EBPβ.

conditions necessary to observe such a multiprotein complex.

]. We have also shown that this same residue is necessary for Ras stimultion of the interaction between C/EBPβ and SRF. Therefore, it will be interesting to determine if this site is also critical for stimulation of the interaction between C/EBPβ and Elk-1. Elk-1 also has several MAPK sites in its C-terminal domain, and therefore this region could also be a target for Ras.

gene in response to mitogenic stimulation.

Conclusions

SRE activation in response to Ras-dependent signaling pathways. We show that SRF, Elk-1, and p35-C/EBPβ are all necessary for maximal Ras-stimulated transactivation of the SRE.

Cell Culture and Transfections

NIH 3T3 fibroblasts (from the American Type Culture Collection) and COS-7 cells (kindly provided by Dr. S. Hann, Vanderbilt University) were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% calf serum (Colorado Serum Company), 0.22% sodium bicarbonate, 4 mM L-glutamine, 25 U of penicillin G sodium per mL, and 25 mg of streptomycin per mL.

-DNA precipitate for 8 h. The medium was removed and replaced with complete medium for 36 h before harvesting. NIH 3T3 transfections were performed using NovaFector (Venn Nova) or Trans-IT LT1 (PanVera) as described by the manufacturers.

]. An internal control plasmid to measure transfection efficiency could not be used because C/EBPβ regulates transcrption from the control plasmid, and thus makes the internal control invalid. Therefore, the transfections were repeated multiple times to control for variability in transfection efficiency.

Plasmids

] into similarly digested pGEX4T-1. pcDNA3/Elk-1(1-209) and pcDNA3/Elk-1(1-140) were constructed by using the Erase-A-Base system (Promega) with pcDNA3/Elk-1 (gift of J. Schwartz, Univ. of Michigan) as described by the manufacturer. pcDNA3.1/His-p35-C/EBPβ was constructed by inserting an 1,739 bp EcoRI fragment from pRSETB-EFII into an EcoRI cut pcDNA3.1/HisC vector (Invitrogen). CMV-LAP was a gift of U. Schibler (Univ. of Geneva, Geneva, Switzerland) and CMV-Ras.V12 was a gift of E. Ruley (Vanderbilt Univ.).

C in PBS containing the protease inhibitors described above. Triton X-100 was added to a final concentration of 0.1%, followed by gentle mixing for 30 min at 4°C. The lysate was clarified at 12,000 × g for 10 min at 4°C. The supernatant was gently mixed with glutathione-sepharose beads (Amersham-Pharmacia) at 4°C for 30 min. Beads containing GST proteins were collected by low speed centrifugation, followed by three successive washes with PBS containing 0.1% Triton X-100 and the protease inhibitors described above.

].

Co-Immunoprecipitation

COS-7 cells were harvested in PBS containing 0.1 mM sodium vanadate and collected by low speed centrifugation. Cells were resuspended in lysis buffer (10 mM Tris (pH 7.5), 1 mM EDTA, 50 mM NaCl, 0.25% Nonidet P-40, 1 mM PMSF, 1 mg of aprotinin/mL, 0.1 mM sodium vanadate, 10 mM sodium molybdate, and 10 mM β-glycerol phosphate) and lysed by sonication with a microtip on setting 2 and 20% duty cycle for 10 s. After clarification by centrifugation at 12,000 × g for 10 min, the cell extract was incubated with T7 tag antibody (Ab)-agarose beads (Novagen) for 2 h. The beads were collected by low speed centrifugation and washed 3 times with lysis buffer. All steps were performed at 4°C. After washing, the beads were boiled for 5 min in Laemmli sample buffer.

Immunoblots

]. A 1:2000 dilution of anti-Elk-1 Ab (Santa Cruz Biotechnology) and a 1:5000 dilution of goat anti-rabbit secondary Ab (Roche Molecular Biochemicals) were used. The secondary Ab was detected using SuperSignal Chemiluminescent Substrate (Pierce).

Abbreviations

, calcium phosphate; C/EBPβ, CCAAT-enhancer binding protein-beta; CAT, chloramphenicol acetyl transferase; DMEM, Dulbecco's Modified Eagle Medium; GST, glutathione-S-transferase; MAPK, mitogen activated protein kinase; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor.

Acknowledgments

We thank John van Doorninick and Michael Hann for expert technical assistance, and Robert Tilghman for critically reviewing the manuscript.

M. Hanlon was supported as a predoctoral trainee on the Cellular, Biochemical, and Molecular Sciences Training Grant 5T32GM08554. L. Bundy was supported by a postdoctoral fellowship from the Molecular Endocrinology Training Grant 5T32DK07563.