Transcriptional Repressor ERF Is a Ras/Mitogen-Activated Protein Kinase Target That Regulates Cellular Proliferation

Abstract

A limited number of transcription factors have been suggested to be regulated directly by Erks within the Ras/mitogen-activated protein kinase signaling pathway. In this paper we demonstrate that ERF, a ubiquitously expressed transcriptional repressor that belongs to the Ets family, is physically associated with and phosphorylated in vitro and in vivo by Erks. This phosphorylation determines the ERF subcellular localization. Upon mitogenic stimulation, ERF is immediately phosphorylated and exported to the cytoplasm. The export is blocked by specific Erk inhibitors and is abolished when residues undergoing phosphorylation are mutated to alanine. Upon growth factor deprivation, ERF is rapidly dephosphorylated and transported back into the nucleus. Phosphorylation-defective ERF mutations suppress Ras-induced tumorigenicity and arrest the cells at the G 0 /G 1 phase of the cell cycle. Our findings strongly suggest that ERF may be important in the control of cellular proliferation during the G 0 /G 1 transition and that it may be one of the effectors in the mammalian Ras signaling pathway.Mitogen-activated protein kinase (MAPK) pathways are a central relay of many extracellular signals leading to change in gene expression. At least three MAPK pathways, which have high structural homology and identity in biochemical mechanisms of activation, have been identified in mammalian cells. The JNK (c-Jun amino-terminal kinase) and p38 pathways are involved primarily in the transduction of stress and cytokine stimuli. The Erk (extracellular signal-regulated kinase) pathway plays a major role in transduction of mitogenic and differentiation stimuli (for reviews, see references 41 and 47). Ras small GTPases have a pivotal role in regulation of proliferation from both receptor tyrosine kinases (RTK) and G proteinmediated receptors, (for reviews, see references 6 and 30). Notably, Ras plays an essential role in the activation of the Raf kinase, which directly phosphorylates and activates the Mek kinase, leading to the activation of Erk1 and Erk2 by phosphorylation on threonine and tyrosine residues. Phosphorylated Erks form homodimers (22) and translocate to the nucleus, where they phosphorylate proteins involved in gene regulation. Besides the Raf/Mek/Erk kinase cascade, other downstream Ras effectors are known to participate in the proliferative response (for a review, see reference 31). For example, phosphoinositide 3-OH kinase (PI3-K) (42) and members of the Rho family (for reviews, see references 17 and 23) have been shown to be responsible for morphological changes induced by Ras and are required for Ras-dependent transformation. Nevertheless, although the implication of Ras pathways, and in particular the Raf/Erk pathway, in the control of proliferation is well established, links with the control of the cell cycle machinery are not clear. Ras-dependent exit from G 0 (45) and progression through G 1 via the control of the retinoblastoma tumor suppressor protein (Rb) (34, 37) have been demonstrated. However, the transcription...

Suppression of the Ewing’s sarcoma phenotype by FLI1/ERF repressor hybrids

“…ERF has been shown to be important for the G 1 transition, and has been shown to enter the nucleus at specific stages of the cell cycle. 24 The FLI1/ERF protein demonstrates a similar mitogen-activated protein kinase-dependent subcellular localization, suggesting that temporary entry of the FLI1/ERF protein into the nucleus is sufficient to suppress the ES phenotype. Because FLI1/ERF and EWS/FLI1 have opposite effects on transcription, the oncogenic activity of the EWS/FLI1 fusion protein is likely to involve activation of genes that can promote or allow inappropriate progression into the G 1 phase of the cell cycle.…”

“…Both inhibition of Erk activity as well as introduction of mutations that affect the ERF subcellular localization can generate a range of repressors with increased nuclear localization and thus increased repressor activity. 24 It has been suggested that "the ideal cancer therapy would accommodate the specific biology of a tumor and be based on understanding the mechanisms of malignancy." 45 In this sense, ERF repressor hybrids, with the unique potential to be regulated both quantitatively and by extracellular signals, may be an approach that could be valuable in the ES system, as well as other systems that involve inappropriate transcription factor activation.…”

“…The 650-bp EcoRI-HindIII fragment of FLI1 was used as the 3Ј-specific probe; the 480-bp BamHI-EcoRI fragment of FLI1 was used as the 5Ј-specific probe. The subcellular localization of the proteins was determined using anti-ERF- 23,24 and anti-FLI1-specific Abs (rabbit polyclonal against the full-length FLI1 protein; D.K.W., unpublished data), 26 as described previously. The electrophoretic mobility shift assay was performed as described previously 23 using protein produced in vitro and the ETS1-3 27 -labeled oligonucleotide as a probe.…”

“…We have shown that the intracellular distribution of ERF is controlled by Erkdependent phosphorylation in response to the proliferation stage of the cell. 24 We examined the subcellular distribution of the FLI1/ERF hybrid and found it to be identical with wt ERF. Thus FLI1/ERF is localized in the cytoplasm of proliferating cells and in the nucleus of serum-arrested cells (Fig 4).…”

“…Furthermore, phosphorylation-deficient ERF mutations can suppress ras-induced tumorigenicity and can arrest cells at the G 0 /G 1 phase of the cell cycle. 24 In this report, we describe our results using retroviral vectors expressing FLI1-ERF hybrid transcriptional repressors to block the transcriptional activation of potential EWS-FLI1 fusion protein targets. Our data indicate both that retroviral gene transfer of a FLI1-ERF hybrid can suppress the ES phenotype and that important specificity elements required for this suppression lie within the ets DNAbinding domain.…”