Due to the common observations that both Elk-1 and p35-C/EBPβ transactivate the SRE, interact with SRF, and are responsive to Ras-dependent signaling pathways, we hypothesized that Elk-1 and p35-C/EBPβ may both be necessary for maximal induction of SRF dependent transcription. To begin to test this hypothesis, we used a GAL4 dependent reporter construct, in which a CAT reporter gene is driven by five copies of a GAL4 binding site upstream of the adenovirus E1B minimal promoter (pG5CAT). We constructed a GAL4 DNA binding domain-SRF fusion protein, as has been described previously [23]. GAL4-SRF binds to the GAL4 reporter construct, thereby making transcription of the reporter gene dependent on the presence of SRF. As shown in Fig. 1, when a GAL4-SRF fusion construct is transfected with the reporter, there is no increase in CAT activity. The SRF transactivation domain is weak, as has been shown previously [24]. 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.

When CMV-LAP (which encodes p35-C/EBPβ) is transfected with GAL4-SRF and the reporter construct, we see a 7-fold increase in CAT activity that is potentiated to 75-fold when activated Ras is cotransfected. Therefore, C/EBPβ results in a much larger increase in transcription than Elk-1. We have previously shown that Ras does not activate the transactivation domain of p35-C/EBPβ [23]. 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.

Since Elk-1 and p35-C/EBPβ synergize in transactivation of SRF-dependent transcription using a GAL4-dependent promoter, we next tested if Elk-1 and p35-C/EBPβ could also synergize in transactivation of a native SRF binding site, namely the c-fos SRE. To test this possibility, NIH 3T3 cells were transiently transfected with a CAT reporter gene driven by one copy of the wild type SRE upstream of the Rous sarcoma virus long terminal repeat minimal promoter. As shown in Fig. 2, 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.

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.

We next tested the possibility that there could be a direct protein-protein interaction between Elk-1 and p35-C/EBPβ based on the fact that they synergize in transactivation of the SRE. Therefore, we used a GST-pulldown assay to determine if the proteins could interact in vitro. p35-C/EBPβ was expressed as a chimeric GST protein and immobilized on glutathione-agarose beads. Beads containing GST-p35-C/EBPβ or GST alone were incubated with in vitro-translated Elk-1 labeled with [³⁵S] methionine. As shown in Fig. 3, lane 2, approximately 35-45% of the input Elk-1 was retained on the GST-p35-C/EBPβ beads. A small amount of Elk-1 bound to the beads containing GST alone (Fig. 3, lane 3) which we have been unable to eliminate even after blocking with unprogrammed translation lysate. However, it is clear that the binding of Elk-1 is substantially increased when the GST-p35-C/EBPβ fusion protein is present on the beads. These data indicate that Elk-1 and p35-C/EBPβ are capable of interacting in vitro.

Since we observed an in vitro interaction between the Elk-1 and C/EBPβ proteins, we next tested whether the proteins could interact in vivo as well. To do this, we used a co-immunoprecipitation approach. COS-7 cells were transfected with an expression vector for a 6X histidine tagged construct of p35-C/EBPβ carrying the φ 10 (T7 tag) epitope sequence either in the presence or absence of Elk-1. Cell lysates were incubated with T7 tag Ab agarose beads followed by immunoblotting of the precipitated proteins with Elk-1 Ab. As shown in Fig. 4, there is no Elk-1 protein precipitated with the tagged C/EBPβ protein when both are transfected (lane 5). However, since the synergism of Elk-1 and p35-C/EBPβ is observed when activated Ras is present, we thought it likely that the interaction between the two proteins could be Ras-dependent. Indeed, when activated Ras is co-transfected along with Elk-1 and histidine-tagged p35-C/EBPβ, the Elk-1 protein is precipitated with the p35-C/EBPβ (lane 6). One possible explanation for this result is that Ras increases the amount of p35-C/EBPβ or Elk-1 protein in the COS-7 cells, but Western blot analysis showed that both p35-C/EBPβ and Elk-1 protein levels are the same in the absence and presence of Ras (data not shown). Therefore, p35-C/EBPβ and Elk-1 interact in vivo, but only in response to activation of Ras-dependent signaling pathways.

Since C/EBPβ and Elk-1 interact, we next wanted to narrow down the domains of the proteins that are required for their interaction. To determine the domain of C/EBPβ that is necessary to interact with Elk-1, a p20-C/EBPβ-GST fusion protein was constructed. p20-C/EBPβ encodes the 20 kDa form of C/EBPβ, which lacks the N-terminal transactivation domain of the longer p35-C/EBPβ isoform. This isoform, however, shares the C-terminal DNA binding and dimerization domain with p35-C/EBPβ. As shown in Fig. 5, approximately the same amount of [³⁵S]-labeled Elk-1 is retained on both the GST-p20-C/EBPβ and GST-p35-C/EBPβ beads (compare lanes 2 and 3). Therefore, deletion of the N-terminus of C/EBPβ has no effect on its ability to interact with Elk-1. This data demonstrates that the C-terminal region of C/EBPβ is sufficient to mediate interaction with Elk-1 in vitro.

In order to narrow down the domain of Elk-1 that is necessary to interact with C/EBPβ in vitro, we made several Elk-1 deletion mutants (Fig. 6A) and tested these constructs in a pulldown assay with GST-p35-C/EBPβ. Elk-1(1-209) is a C-terminal deletion mutant that lacks the C-box. Elk-1(1-140) is also a C-terminal deletion, but it lacks both the B- and C-boxes. Therefore, this mutant contains neither a transactivation domain nor an SRF binding domain. Finally, Elk-1(89-428) is a deletion of the N-terminal A-box. Glutathione agarose beads containing GST-p35-C/EBPβ or GST alone were incubated with in vitro-translated Elk-1 mutants labeled with [³⁵S] methionine. As shown in Fig. 6B, the C-terminal Elk-1 mutants, Elk-1(1-209) and Elk-1(1-140), bound to the GST-p35-C/EBPβ beads to the same extent as wild-type Elk-1 (compare lane 3 of top 3 panels). However, the A-box deletion mutant, Elk-1(89-428), no longer binds to the GST-p35-C/EBPβ (lane 3, bottom panel). Therefore, this data demonstrates that the A-box of Elk-1 is necessary to interact with C/EBPβ in vitro.

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.

COS-7 cell transfections were performed by the calcium phosphate (CaPO₄) technique [31]. Cells at 60-70% confluence were exposed to the CaPO₄-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.

For NovaFector transfections, a 6:1 ratio of NovaFector:DNA was used for each transfection. Cells at 60-70% confluence were exposed to the NovaFector:DNA complex for 6-7 h in serum and antibiotic free medium. After the incubation, an equal volume of DMEM containing 20% calf serum was added for 24 h, bringing the final concentration of calf serum to 10%. Cells were then serum deprived for 36-40 h in DMEM supplemented as above except containing 0.5% calf serum before harvesting. For TransIT-LT1 transfections, a 6:1 ratio of TransIT-LT1:DNA was used in each transfection. Cells at 60-70% confluence were exposed to the TransIT-LT1:DNA complex for 8 h in complete medium plus freshly added 30 mg/mL polymyxin B antibiotic [32]. The medium was then removed and replaced with complete medium for 24 h. Cells were then serum deprived in DMEM containing 0.5% calf serum for 36-40 h before harvesting. Cell extracts were prepared and chloramphenicol acetyl transferase (CAT) assays were performed on extracts containing equivalent cell protein as previously described [33]. 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.

The pGAL4 and pG5CAT plasmids were obtained from the Mammalian MATCHMAKER Two-Hybrid Assay Kit (CLONTECH). pGAL4-SRF was constructed as previously described [23]. The SRE-CAT reporter gene was constructed as previously described [22]. pGST-p35-C/EBPβ was constructed by inserting the 1,739 bp EcoRI fragment from pRSETB-EFII [34] into EcoRI cut pGEX-4T-1 (Amersham-Pharmacia). pGST-p20-C/EBPβ was constructed by inserting the 581 bp BamHI/EcoRI fragment from pRsetA-LIP [34] 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.).

GST-p35-C/EBPβ, GST-p20-C/EBPβ, or GST alone was produced in BL21 E. Coli by 80 mM isopropyl-β-D-thiogalactopyranoside (IPTG) induction overnight at 37°C. Cells were harvested by centrifugation and washed once with phosphate-buffered saline (PBS) containing the following protease inhibitors: 2 mg/ml aprotinin, 2 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.2 mM pepstatin. The bacteria were lysed by sonication at 4^oC 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.

The ELK-1, ELK-1(1-209), ELK-1(1-140), and ELK-1(89-428) proteins were transcribed and translated in vitro using the TNT T7 coupled reticulocyte lysate system (Promega) according to the manufacture's instructions. Translated proteins were radiolabeled with the EXPRE³⁵S³⁵S protein labeling mix (Dupont NEN). The GST-pulldown assay was done as previously described [22].

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.

The samples from the coimmunoprecipitations described above were analyzed by electrophoresis on an SDS-12% polyacrylamide gel for 3 h at 160 V. The gel was equilibrated in transfer buffer (33 mM Tris base, 192 mM glycine, 20% methanol) for 15 min before transfer of proteins to Immobilon-P membrane (Millipore Corp.). After transfer, an immunoblot was performed as described previously [22]. 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).