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...

The ets domain transcriptional repressor ERF is an effector of the receptor tyrosine kinase/Ras/Erk pathway, which, it has been suggested, is regulated by subcellular localization as a result of Erk-dependent phosphorylation and is capable of suppressing cell proliferation and ras-induced tumorigenicity. Here, we analyze the effect of ERF phosphorylation on nuclear import and export, the timing of its phosphorylation and dephosphorylation in relation to its subcellular location, Erk activity, and the requirements for ERF-induced cell cycle arrest. Our findings indicate that ERF continuously shuttles between the nucleus and the cytoplasm and that both phosphorylation and dephosphorylation of ERF occur within the nucleus. While nuclear import is not affected by phosphorylation, ERF nuclear export and cytoplasmic release require multisite phosphorylation and dephosphorylation. ERF export is CRM1 dependent, although ERF does not have a detectable nuclear export signal. ERF phosphorylation and export correlate with the levels of nuclear Erk activity. The cell cycle arrest induced by nonphosphorylated ERF requires the wild-type retinoblastoma protein and can be suppressed by overexpression of cyclin. These data suggest that ERF may be a very sensitive and constant sensor of Erk activity that can affect cell cycle progression through G 1 , providing another link between the Ras/Erk pathway and cellular proliferation.

1 The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44-and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2 To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Go¨6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Go¨6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (e, d), but not conventional or atypical PKCs. Further analyses suggest that PKCe is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC-and PKCe-dependent pathway.

The ERF transcriptional repressor is a downstream effector of the RAS/ERK pathway that interacts with and is directly phosphorylated by ERKs ETS2-repressor factor (ERF)3 is a ubiquitously expressed transcriptional regulator of the ETS family of transcription factors, with tumor suppressor activity, that is regulated by the RAS/ERK signaling pathway. ERF is shown to be bound and phosphorylated both in vivo and in vitro by ERKs (1, 2). It interacts specifically with active and inactive ERKs via two distinct FXF motifs and can effectively block ERK-substrate interaction (3). In the absence of growth factors, ERF is dephosphorylated and located in the nucleus, whereas upon mitogenic stimulation and in exponentially growing cells, it is actively transported into the cytoplasm through a CRM-dependent mechanism (4). Phosphorylation-deficient ERF mutants are able to reverse RAS-induced tumorigenicity and arrest fibroblasts in the G 0 /G 1 phase of the cell cycle, determining ERF as a bona fide ERK substrate and an effector of the RAS/ERK pathway (2-4). ERF-mediated cell cycle arrest can be abolished by the overexpression of cyclins D and E or the inactivation of the retinoblastoma protein, providing a strong link with cell cycle regulation (2, 4). Homozygous deletion of Erf leads to a block of chorionic trophoblast differentiation, the absence of chorioallantoic fusion, persisting chorion layer, the absence of labyrinth formation, expansion of the giant cell layer, diminishing of the spongiotrophoblast layer, and eventual embryo death by 10.5 dpc (5). Trophoblast stem cell lines derived by Erf Ϫ/Ϫ embryos exhibit delayed differentiation kinetics and decreased expression of spongiotrophoblast terminal differentiation markers suggesting that the ERF is required for extraembryonic ectoderm and trophoblast stem cell differentiation. Thus, there is emerging evidence for ERF contribution in cell cycle inhibition and promotion of differentiation. However, relevant downstream ERF targets have not yet been identified, rendering unclear its mechanism of action.c-MYC is a ubiquitously expressed transcription factor that in physiological levels binds about 10% of the human promoters (6) and regulates crucial cell functions such as proliferation, differentiation, apoptosis, metabolism, and cell growth (for review see Ref. 7). c-MYC induces the activity of cyclin-cyclindependent kinase complexes or affects directly the expression of cell cycle regulators (8 -15), suggestive of its role in cell cycle progression and consistent with the severely retarded proliferation of the c-Myc Ϫ/Ϫ primary mouse fibroblasts because of G 1 and G 2 phase lengthening (16,17). Heterozygous c-Myc fibroblasts show slower growth rates (18), whereas c-MYC controls mammalian and fly body size in a dose-dependent manner (19 -21), indicating that subtle perturbations in c-MYC levels lead to profound defects in cell and organismal physiology.Tight regulation of c-Myc expression is achieved in transcriptional, post-transcriptional, translational, and post-transl...

Altogether these results strongly suggest the involvement of the proteasome system in the pathogenesis of psoriasis and support the relevance of proteasome inhibitors in local or systemic treatment of psoriasis.

Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway.

Fusion of the 5Ј half of the Ewing's sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing's phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation. A berrant expression and/or structural alteration of transcription factors have been suggested to be critical events in tumorigenic transformation. 1,2 The t(11;22)(q24;q12) chromosomal translocation, detected in the majority of Ewing's sarcoma (ES) and primitive neuroectodermal tumors, fuses the amino terminus of the EWS gene to the carboxyl terminus of the ets transcription factor family member FLI1. 3 In other ES and primitive neuroectodermal tumors, analogous translocations fuse the EWS gene to other members of the ets family, including ERG, 4 ETV-1, 5 E1AF, 6 and FEV, 7 members of the ERG and PEA3 subclasses of the family. 8,9 In all of these fusions, the transactivating domain of the EWS gene is fused to the DNA-binding domain of an ets gene. It has been shown that the EWS/FLI1 fusion protein is a more potent transcriptional activator than FLI1 10,11 and, in contrast to FLI1, is a transforming gene. 10 Both the transactivation domain of EWS and the ets-DNA-binding domain are required for transformation, 10 and the transactivation efficiency of different classes of EWS-FLI1 fusions has been reported to correlate with the behavior of tumors in vivo. 12 Furthermore, the transforming phenotype can be suppressed by blocking EWS-FLI1 production. [13][14][15] The parental EWS protein contains an RNA-binding domain and a transactivation domain and is similar to the TLS/FUS and hTAF II 68 members of the TET family of proteins that are able to interact with both TFIID and RNA polymerase II. 16 The EWS-FLI1 hybrid, however, does not appear to be capable of similar functions, 17 suggesting that different mechanisms may be involved in EWS-FLI1-induced tumorigenesis.The...

Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha). Therefore, we generated HaCaT cells that stably express human MC1R or the Arg151Cys (R151C) nonfunctional variant. We demonstrate that: (1) the constitutive activity of MC1R results in elevated intracellular cAMP level, reduced NF-kappaB activity and decreased TNF-alpha transcription; (2) binding of alpha-MSH to MC1R and the subsequent increase in cAMP production do not inhibit TNFalpha-mediated NF-kappaB activation; (3) MC1R signaling is sufficient to strongly inhibit UVB-induced TNF-alpha expression and this inhibitory effect is further enhanced by alpha-MSH stimulation. Our findings suggest that the constitutive activity of the G-protein-coupled MC1R in keratinocytes may contribute to the modulation of inflammatory events and immune response induced by UV light.