The ternary complex factor (TCF) subfamily of ETS-domain transcription factors form ternary complexes with the serum response factor (SRF) and the c-fos SRE. Extracellular signals are relayed via MAP kinase signal transduction pathways through the TCF component of the ternary complex. Protein-protein interactions between TCFs and SRF play an essential role in formation of this ternary complex. A 30 amino acid sequence encompassing the TCF B-box is sufficient to mediate interactions with SRF. In this study we have identified amino acids which are critical for this interaction and derived a molecular model of the SRF binding interface. Alanine scanning of the Elk-1 B-box reveals five predominantly hydrophobic residues which are essential for binding to SRF and for ternary complex formation in vitro and in vivo. These amino acids are predicted to lie on one face of an alpha-helix. Peptides encompassing the B-box retain biological activity and have helix-forming propensity. alpha-Helix and ternary complex formation is disrupted by the introduction of helix-breaking proline residues. Our results are consistent with a model in which the Elk-1 B-box forms an inducible alpha-helix which presents a hydrophobic face for interaction with SRF. We discuss the wider applicability of our results to similar short protein-protein interaction motifs found in other transcription factors.

The Elk-1 transcription factor is a member of a group of proteins called ternary complex factors, which serve as a paradigm for gene regulation in response to extracellular signals. Its deregulation has been linked to multiple human diseases including the development of tumours. The work herein aims to inform the design of potential peptidomimetic compounds that can inhibit the formation of the Elk-1 dimer, which is key to Elk-1 stability. We have conducted molecular dynamics simulations of the Elk-1 ETS domain followed by virtual screening.

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

Elk-1, a member of the ternary complex factors (TCFs) within the ETS (E26 transformation-specific) domain superfamily, is a transcription factor implicated in neuroprotection, neurodegeneration, and brain tumor proliferation. Except for known targets, c-fos and egr-1, few targets of Elk-1 have been identified. Interestingly, SMN, SOD1, and PSEN1 promoters were shown to be regulated by Elk-1. On the other hand, Elk-1 was shown to regulate the CD133 gene, which is highly expressed in brain-tumor-initiating cells (BTICs) and used as a marker for separating this cancer stem cell population. In this study, we have carried out microarray analysis in SH-SY5Y cells overexpressing Elk-1-VP16, which has revealed a large number of genes significantly regulated by Elk-1 that function in nervous system development, embryonic development, pluripotency, apoptosis, survival, and proliferation. Among these, we have shown that genes related to pluripotency, such as Sox2, Nanog, and Oct4, were indeed regulated by Elk-1, and in the context of brain tumors, we further showed that Elk-1 overexpression in CD133+ BTIC population results in the upregulation of these genes. When Elk-1 expression is silenced, the expression of these stemness genes is decreased. We propose that Elk-1 is a transcription factor upstream of these genes, regulating the self-renewal of CD133+ BTICs.

The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.

Serum stimulation of mammalian cells induces, via the MAPK pathway, the nuclear protein DUSP5 (dual-specificity phosphatase 5), which specifically interacts with and inactivates the ERK1/2 MAP kinases. However, molecular mechanisms underlying DUSP5 induction are not well known. Here, we found that the DUSP5 mRNA induction depends on a transcriptional regulation by the MAPK pathway, without any modification of the mRNA stability. Two contiguous CArG boxes that bind serum response factor (SRF) were found in a 1 Kb promoter region, as well as several E twenty-six transcription factor family binding sites (EBS). These sites potentially bind Elk-1, a transcription factor activated by ERK1/2. Using wild type or mutated DUSP5 promoter reporters, we demonstrated that SRF plays a crucial role in serum induction of DUSP5 promoter activity, the proximal CArG box being important for SRF binding in vitro and in living cells. Moreover, in vitro and in vivo binding data of Elk-1 to the same promoter region further demonstrate a role for Elk-1 in the transcriptional regulation of DUSP5. SRF and Elk-1 form a ternary complex (Elk-1-SRF-DNA) on DUSP5 promoter, consequently providing a link to an important negative feedback tightly regulating phosphorylated ERK levels.

Pea3 proteins belong to a subfamily of the E-twentysix (ETS) domain superfamily of transcription factors, which play various roles during development. Polyoma Enhancer-Activator 3 (Pea3) proteins Pea3, ERM and Er81 are particularly involved in tissues with branching morphogenesis, including kidney, lung, mammary gland and nervous system development. A recent transcriptomic study on novel targets of Pea3 transcription factor revealed various axon guidance and nervous system development related targets, supporting a role of Pea3 proteins in motor neuron connectivity, as well as novel targets in signaling pathways involved in synaptic plasticity. This study focuses on the expression of Pea3 family members in hippocampal neurons, and regulation of putative Pea3 targets in Pea3-overexpressing cell lines and following induction of long-term potentiation or seizure in vivo. We show that Pea3 proteins are expressed in hippocampus in both neuronal and non-neuronal cells, and that Pea3 represses Elk-1 but activates Prkca and Nrcam expression in hippocampal cell lines. We also show that mRNA and protein levels of Pea3 family members are differentially regulated in the dentate gyrus and CA1 region upon MECS stimulation, but not upon LTP induction.

Although microRNAs (miRNAs or miRs) are able to function as oncogenes or tumor suppressors, the role of miR-326 in regulating human cervical cancer cells remains unclear. In the present study, the expression of miR-326 was identified to be downregulated in cervical cancer cell lines and primary tumor samples, and the overexpression of miR-326 decreased cell proliferation, migration and invasion in cervical cell lines. Bioinformatics prediction and experimental validation results revealed that the function of miR-326 was achieved by targeting and repressing ETS domain-containing protein Elk-1 (ELK1) expression. ELK1 was targeted directly by miR-326, which was downregulated in human cervical cancer tissues compared with that in adjacent normal tissues. The results of the present study suggest that miR-326, a potential tumor suppressor, may be used in the treatment of cervical cancer.

The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.

Cerebral vasospasm (CVS) is a common complication of subarachnoid hemorrhage (SAH) with high deformity rates and cerebral vascular smooth muscle cells (VSMCs) phenotypic switch is considered to be involved in the regulation of CVS. However, to the best of the authors' knowledge, its underlying molecular mechanism remains to be elucidated. Peroxisome proliferator‑activated receptor β/δ (PPARβ/δ) has been demonstrated to be involved in the modulation of vascular cells proliferation and maintains the autoregulation function of blood vessels. The present study investigated the potential effect of PPARβ/δ on CVS following SAH. A model of SAH was established by endovascular perforation on male adult Sprague‑Dawley rats, and the adenovirus PPARβ/δ (Ad‑PPARβ/δ) was injected via intracerebroventricular administration prior to SAH. The expression levels of phenotypic markers α‑smooth muscle actin and embryonic smooth muscle myosin heavy chain were measured via western blotting or immunofluorescence staining. The basilar artery diameter and vessel wall thickness were evaluated under fluorescence microscopy. SAH grade, neurological scores, brain water content and brain swelling were measured to study the mechanisms of PPARβ/δ on vascular smooth muscle phenotypic transformation. It was revealed that the expression levels of synthetic proteins were upregulated in rats with SAH and this was accompanied by CVS. Activation of PPARβ/δ using Ad‑PPARβ/δ markedly upregulated the contractile proteins elevation, restrained the synthetic proteins expression and attenuated SAH‑induced CVS by regulating the phenotypic switch in VSMCs at 72 h following SAH. Furthermore, the preliminary study demonstrated that PPARβ/δ downregulated ERK activity and decreased the expression of phosphorylated (p‑)ETS domain‑containing protein Elk‑1 and p‑p90 ribosomal S6 kinase, which have been demonstrated to serve an important role in VSMC phenotypic change. Additionally, it was revealed that Ad‑PPARβ/δ could positively improve CVS by ameliorating the diameter of the basilar artery and mitigating the thickness of the vascular wall. Furthermore, subsequent experiments demonstrated that Ad‑PPARβ/δ markedly reduced the brain water content and brain swelling and improved the neurological outcome. Taken together, the present study identified PPARβ/δ as a useful regulator for the VSMCs phenotypic switch and attenuating CVS following SAH, thereby providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

RAS oncogene family members are molecular switches of signaling pathways that control cell growth, proliferation, differentiation, and survival. In colorectal cancer, Kirsten-RAS (KRAS) and neuroblastoma-RAS (NRAS) are the commonly mutated isoforms. Activating mutations in RAS result in cellular transformation independent of upregulated epidermal growth factor receptor (EGFR)-initiated signaling. The present study characterized the functional consequences of non-canonical/novel KRAS and NRAS mutants identified in a targeted next-generation sequencing study of colorectal cancer specimens from Filipino patients. In vitro assays in NIH3T3 cells showed that similar to the canonical KRAS G12D mutant, overexpression of KRAS G12S, A59T, and Y137C, but not NRAS G12D and NRAS A11V, confer higher proliferation and migration rates. HCT116 cells transfected with the novel NRAS A11V and the canonical NRAS G12D, but not the KRAS mutants, display enhanced resistance to apoptosis. All four non-canonical/novel KRAS and NRAS mutants induce gross changes in F-actin cytoskeletal organization and cellular morphology of NIH3T3 cells. Only KRAS G12S and KRAS A59T appear to deregulate extracellular signal-regulated kinase (ERK) and its downstream target ETS transcription factor ELK1 (ELK1). Elucidation of differential effector engagement responsible for the variable phenotypic readouts of the mutants is warranted. If validated by mouse studies and clinical correlates, these can have wider implications in choosing treatment options.