hTERT Is highly expressed in Ewing's sarcoma and activated by EWS-ETS oncoproteins.

Although the fusion proteins EWS-ETS are uniquely associated with Ewing's sarcoma and have been shown to have transformational properties, they are not the only determinants of oncogenesis. Therefore, before molecular-based therapy can be initiated, a better understanding of the molecular network specific to Ewing's sarcoma is mandatory. Specimens from 31 patients with Ewing's sarcoma were analyzed immunohistochemically. We found that human telomerase reverse transcriptase was expressed highly (78%) in Ewing's sarcoma. The mean followup was 7 years (range, 1-21 years), and human telomerase reverse transcriptase expression was correlated with outcome. Because we did not find an association between expression pattern and survival, human telomerase reverse transcriptase may not serve as a tumor marker in Ewing's sarcoma. However, the human telomerase reverse transcriptase promoter is shown to be activated by the fusion proteins. Therefore, transcriptional regulation via EWS-ETS may account for the high human telomerase reverse transcriptase expression in Ewing's sarcoma.

The versatile functions of the transcriptional coactivators p300 and CBP and their roles in disease.

p300 and CBP are highly homologous coactivators which promote gene transcription by bridging between DNA-binding transcription factors and the basal transcription machinery, by providing a scaffold for integrating transcription factors, and by modifying transcription factors and chromatin through acetylation. The p300/CBP cofactors are involved in a plethora of physiological processes, and their activity is essential for embryogenesis. Chromosomal translocations affecting the p300 and Cbp genes are the cause of hematological malignancies, and Cbp haploinsufficiency is a hallmark of the Rubinstein-Taybi syndrome. In addition, mutations in the Cbp or p300 gene, accompanied by loss of the other allele, have been found in various kinds of tumors. Furthermore, inhibition of CBP and p300 function in neurodegenerative diseases caused by polyglutamine expansion may be an underlying cause for cytotoxicity. Approaches to modulate p300/CBP function may be instrumental in the development of novel therapies directed against viral infections, cancer and neurodegenerative diseases.

HER2/Neu-mediated activation of the ETS transcription factor ER81 and its target gene MMP-1.

In this study, we show that the ETS transcription factor ER81 directly binds to and activates the promoter of the matrix metalloproteinase gene, MMP-1. Further, the oncoprotein HER2/Neu synergizes with ER81 to stimulate MMP-1 transcription. The activation of ER81 by HER2/Neu is mediated by MAP kinases, which phosphorylate ER81 in its N-terminal activation domain. Four respective phosphorylation sites have been identified. Blocking phosphorylation at these sites decreases ER81 transcriptional activity, which can be further diminished by abolishment of phosphorylation at two non-MAP kinase sites. Altogether, our results reveal mechanisms of how phosphorylation of ER81 regulates the expression of target genes such as MMP-1, which may be important for many physiological processes from embryogenesis to adulthood as well as for tumor metastasis.

Cell type-specific inhibition of the ETS transcription factor ER81 by mitogen-activated protein kinase-activated protein kinase 2.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is an important intracellular mediator of stress signals. In this report, a novel target of MK2 has been identified, the ETS transcription factor family member ER81, whose dysregulation contributes to tumorigenesis and whose normal function is required during development. MK2 phosphorylates ER81 in vitro within its central inhibitory domain, and overexpression of MK2 leads to increased in vivo phosphorylation of ER81. Two serine residues, ER81 amino acids 191 and 216, were identified as MK2 phosphorylation sites. MK2 suppresses basal ER81-dependent transcription, and this suppressive effect is alleviated upon mutation of the MK2 phosphorylation sites in a cell type-specific manner. However, MK2 can also interfere with ER81-mediated transcription independently of serine 191 and serine 216 phosphorylation. Furthermore, MK2 overexpression counteracts the stimulation of ER81 activity by p38 mitogen-activated protein kinase. Altogether, MK2 may regulate ER81 transcriptional activity in a cell type-specific manner and thereby modulate various physiological processes beyond stress responses.

The Ewing's sarcoma gene product functions as a transcriptional activator.

The Ewing's sarcoma (EWS) proto-oncogene can give rise to a variety of different tumors because of the generation of transforming EWS fusion proteins upon chromosomal translocation. However, the cellular function of the EWS protein itself was hitherto not established. We show that EWS is a nuclear protein, whose nuclear localization is dependent upon its transactivating NH2 terminus. EWS COOH-terminal amino acids suppress this NH2-terminal activation domain in the context of a Gal4 fusion protein, which may explain why none of the EWS fusion proteins in cancer cells contains the EWS COOH terminus. Furthermore, EWS expression enhances c-fos, Xvent-2, and ErbB2 promoter activity in a cell-type-dependent manner, indicating that EWS is a transcriptional regulator. Also, the EWS protein stimulates transcription mediated by the COOH-terminal transactivation domain of the cofactor CREB-binding protein (CBP). Coimmunoprecipitation experiments demonstrate that EWS forms a complex with CBP and the homologous p300 protein. A COOH-terminal region of EWS is both required for the physical interaction with CBP/p300 and sufficient to mediate c-fos activation. In addition, suppression of CBP/p300 function by the adenoviral E1A protein abolishes c-fos activation by EWS, indicating that EWS-mediated gene regulation depends on CBP/p300. In conclusion, the nuclear EWS proto-oncoprotein can function as a transcriptional cofactor in conjunction with CBP/p300.

Phosphorylation of ETS transcription factor ER81 in a complex with its coactivators CREB-binding protein and p300.

The ETS protein ER81 is a DNA-binding factor capable of enhancing gene transcription and is implicated in cellular transformation, but presently the mechanisms of its actions are unclear. In this report, ER81 is shown to coimmunoprecipitate with the transcriptional coactivator CREB-binding protein (CBP) and the related p300 protein (together referred to as CBP/p300). Moreover, confocal laser microscopic studies demonstrated that ER81 and p300 colocalized to nuclear speckles. In vitro and in vivo interaction studies revealed that ER81 amino acids 249 to 429, which encompass the ETS DNA-binding domain, are responsible for binding to CBP/p300. However, mutation of a putative protein-protein interaction motif, LXXLL, in the ETS domain of ER81 did not affect interaction with CBP/p300, whereas DNA binding of ER81 was abolished. Furthermore, two regions within CBP, amino acids 451 to 721 and 1891 to 2175, are capable of binding to ER81. Consistent with the physical interaction between ER81 and the coactivators CBP and p300, ER81 transcriptional activity was potentiated by CBP/p300 overexpression. Moreover, an ER81-associated protein kinase activity was enhanced upon p300 overexpression. This protein kinase phosphorylates ER81 on serines 191 and 216, and mutation of these phosphorylation sites increased ER81 transcriptional activity in Mv1Lu cells but not in HeLa cells. Altogether, our data elucidate the mechanism of how ER81 regulates gene transcription, through interaction with the coactivators CBP and p300 and an associated kinase that may cell type specifically modulate the ability of ER81 to activate gene transcription.

CBP/p300 interact with and function as transcriptional coactivators of BRCA1.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor, with properties of a transcription factor involved in DNA repair. We previously have shown the transactivation of heterologous promoters by the carboxyl terminus of BRCA1. We now describe that BRCA1-mediated transactivation is enhanced by p300/CBP (CREB binding protein) and that this effect was suppressed by the adenovirus E1A oncoprotein. We show a physical association of BRCA1 with the transcriptional coactivators/acetyltransferases p300 and CBP. Endogenous as well as overexpressed BRCA1 and p300 were found to associate in a phosphorylation-independent manner. BRCA1 interacts with the cAMP response element binding protein (CREB) domain of p300/CBP via both its amino and carboxyl termini. Finally, full-length BRCA1 is shown to transcriptionally activate the Rous sarcoma virus-long terminal repeat promoter, which was further stimulated by p300. Immunocolocalization analyses suggest that BRCA1 and p300 associate in a cell cycle-dependent manner. Our results support a role for BRCA1 in transcription.

Role of focal adhesion kinase in MAP kinase activation by insulin-like growth factor-I or insulin.

Integrin-induced focal adhesion kinase (FAK) phosphorylation as well as insulin-like growth factor-I (IGF-I) and insulin activate MAP kinase. Since IGF-I or insulin have been suggested to affect FAK phosphorylation, we analyzed the role of FAK in IGF-I- or insulin-induced MAP kinase activation. Although MAP kinase was stimulated by IGF-I or insulin, FAK tyrosine phosphorylation remained unchanged in fibroblasts expressing normal or transiently elevated levels of IGF-I and insulin receptors. Further analysis in FAK deficient fibroblasts suggested that FAK impedes MAP kinase activation by IGF-I or insulin.

Activation of Smad1-mediated transcription by p300/CBP.

Smad1 is the intracellular effector of bone morphogenetic proteins (BMPs), a growth factor family well known to stimulate bone and cartilage formation. Smad1 becomes phosphorylated by the cognate BMP transmembrane receptor protein kinases, associates with the common mediator Smad4 and translocates to the nucleus where it is involved in regulation of gene transcription. In this report we demonstrate that Smad1 interacts with the paralogous coactivators p300 and CBP both in vitro and in vivo. The N- and C-termini of Smad1 negatively interfere with binding to p300/CBP, and the C-terminal half of Smad1 harbors two interaction domains both binding to the same region near the C-terminus of p300/CBP. We show that Smad1 as well as a Gal4 fusion with the C-terminal half of Smad1 stimulate gene transcription in a cooperative fashion with p300/CBP. Phosphorylation of Smad1 enhances its binding to CBP and thereby further stimulates Smad1-dependent transcription. These results provide a mechanism how phosphorylated Smad1 regulates gene activity by interaction with p300/CBP, and factors associated with these bridging coactivators.

TGF-beta-stimulated cooperation of smad proteins with the coactivators CBP/p300.

TGF-beta and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. We report that TGF-beta receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 is negatively regulated by Smad-intramolecular interactions. Furthermore, p300 and TGF-beta receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-beta signaling and may mediate the antioncogenic functions of Smad2 and Smad4.

The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136.

The c-kit-encoded receptor protein tyrosine kinase for stem cell factor (Kit/SCF-R) is essential for the development of cells within the hematopoietic, melanogenic and gametogenic lineages. SCF stimulation induces activation of phosphatidylinositol (PI) 3-kinase, which is required for SCF-induced mitogenesis and cell survival, and for activation of the serine/threonine, we found that, in response to SCF Akt became activated and mediated phosphorylation of Bad, a pro-apoptotic molecule, in a PI-3-kinase-dependent manner. Phosphorylation of Bad was restricted to Ser112 and Ser136 in vivo, but only the Akt phosphorylation sit Ser136 was essential for SCF-promoted cell survival. Furthermore, Bad and Akt interacted and colocalized in intact cells. A Kit/SCF-R gain-of-function mutant that has increased mitogenic and PI 3-kinase activation potential, due to the absence of the two protein kinase C negative feedback phosphorylation site, enhanced both Akt activation and Bad phosphorylation and also resulted in increased cell survival. Such a mechanism may account for how deregulated PI 3-kinase activity and naturally occurring gain-of-function point mutants of Kit/SCF-R lead to cellular transformation and fatal malignancies in man.

p38-2, a novel mitogen-activated protein kinase with distinct properties.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

Quantification of additional short arms of chromosome 12 in germ cell tumours using the polymerase chain reaction.

Male germ cell tumours are characterised by the over-representation of 12p sequences, most often in the form of isochromosome i(12p). This study describes the development of a quantitative detection system for additional copies of 12p employing the polymerase chain reaction (PCR). The validity of this method was assessed on two i(12p) containing tumour cell lines in which the number of i(12p) was determined by fluorescence in situ hybridisation. Fourteen primary male germ cell tumours were analysed using the PCR-based method. While 3/8 seminomatous germ cell cancers did not contain any additional 12p, all 6 non-seminomatous tumours did and the severity of the disease correlated with the respective copy number. The ease of the PCR-based method makes it possible for the quantification of additional 12p to become a routine diagnostic and prognostic tool for testicular germ cell tumours, thereby helping to define the role of the i(12p) anomality in larger retrospective studies.

Transcriptional activity and constitutive nuclear localization of the ETS protein Elf-1.

Elf-1 is a lymphoid-specific transcription factor that belongs to the ETS protein family. It can bind to DNA target sequences within a variety of cytokine genes. We demonstrate that Elf-1 is constitutively localized in the nucleus which is dependent on the presence of amino acids 86-265. Analysis of Gal4-Elf-1 fusion proteins revealed that the N-terminal 86 amino acids of Elf-1 contain a transcriptional activation domain, the activity of which is attenuated by an internal repression domain. Furthermore, Elf-1 interacts specifically with the E74 target sequence and can stimulate transcription driven by the E74 site independent of mitogenic signaling. Thus, Elf-1 is able to stimulate gene transcription which may be required for the development and activity of lymphocytes.

Convergence of MAP kinase pathways on the ternary complex factor Sap-1a.

The serum response element (SRE), which is pivotal for transcriptional up-regulation of the c-fos protooncogene, is constitutively occupied by a protein complex comprising the serum response factor and a ternary complex factor (TCF). Phosphorylation of the TCFs Elk-1 and Sap-1a by the ERK and JNK subclasses of MAP kinases triggers c-fos transcription. We demonstrate here that Elk-1 is barely activated by a third subclass of MAP kinases (p38), most likely because the critical residues Ser383 and Ser389 are poorly phosphorylated by p38 MAP kinase. In contrast, the TCF Sap-1a is efficiently phosphorylated by p38 MAP kinase in vitro and in vivo on the homologous residues Ser381 and Ser387. Mutation of these sites to alanine severely reduces c-fos SRE-dependent transcription mediated by Sap-1a and p38 MAP kinase. Thus, Sap-1a may be an important target for mitogens, stress and apoptotic signals to elicit a nuclear response. However, signaling from p38 MAP kinase to Sap-1a or from Sap-1a to the basal transcription machinery does not occur in all cell types nor at promoters other than the c-fos SRE, which may ensure the specificity of signaling.

Activation of the Sap-1a transcription factor by the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase.

Ternary complex factors (TCFs) bind to the serum response element in the c-fos promoter and mediate its activation by many extracellular stimuli. Some of these stimuli activate the ERK subclass of mitogen-activated protein kinases (MAPKs) that target the TCF Sap-1a. We show that Sap-1a is also phosphorylated by the stress-activated JNK subclass of MAPKs leading to stimulation of both c-fos serum response element and E74-site-dependent transcription in RK13 cells. Several JNK-1 phosphorylation sites were mapped within Sap-1a, and mutation of these sites affected the transactivation mediated by Sap-1a and JNK-1. The impact of these phosphorylation sites varied at different promoters and was dependent on whether Sap-1a was stimulated by ERK-1 or JNK-1. Additionally, a comparison of Sap-1a with another TCF, Elk-1, revealed that these proteins behaved differently to stimulation by ERK-1 and JNK-1. Furthermore, activation of Sap-1a by JNK-1 was inhibited by the p38(MAPK) in RK13 cells, possibly by competition for a common upstream activator. Altogether, our data suggest that Sap-1a plays an important role in the nuclear response elicited by cellular stress.

MAP kinase-dependent transcriptional coactivation by Elk-1 and its cofactor CBP.

A plethora of signals induce the c-fos proto-oncogene via phosphorylation of the transcription factor Elk-1 by MAP kinase. We show that the coactivator CBP cooperates with Elk-1 to stimulate c-fos. Elk-1 physically interacts with CBP, which is dependent on the transactivation domain of Elk-1 but is independent of MAP kinase phosphorylation. However, functional cooperation between Elk-1 and CBP requires phosphorylation of Elk-1. Importantly, a carboxy-terminal transactivation domain of CBP itself is phosphorylated by MAP kinase, whereby the transactivation potential of CBP is enhanced. Thus, MAP kinase may not solely activate specific transcription factors but also the coactivator CBP, identifying a novel aspect of MAP kinase function. Thereby MAP kinase stimulation can pleiotropically affect activation of genes regulated by different transcription factors interacting with the same coactivator CBP.

The ETS-related transcription factor ERM is a nuclear target of signaling cascades involving MAPK and PKA.

Recent studies support a model for signal transduction from activated receptor tyrosine kinases to Ras which, in turn, activates the pathway of the mitogen-activated protein kinase (MAPK). Although some members of the Ets transcription factor family have been shown to be activated by this signaling pathway, no data are available on the activation of the PEA3 group of Ets proteins. This group is composed of three members -- PEA3, ER81 and ERM -- which are very similar in the DNA-binding domain, the ETS domain, in the 32 residue amino-terminal acidic domain and in the 61 residue carboxy-terminal domain. First of all we demonstrated that ERM-transfected cells contain a positive labeling in the nucleus, and we concluded that a nuclear localization signal might be situated in the ETS domain. We then showed that of four putative reporter plasmids, ERM activated the artificial 3 x TORU plasmid which contains an Ets binding site contiguous to an AP1 one. This transactivation enhancement requires the presence of the ERM amino-terminal domain. In contrast, although the lack of the carboxy-terminal domain induced a decrease in transactivation, this latter domain is not crucial. By using the E74-reporter plasmid system which is not basically activated by ERM, we showed that the activation of the Ras/Raf-1/MAPK pathway significantly enhanced ERM-mediated transactivation. The deletion of the amino-terminal transactivation domain abolished the capacity of stimulated MAPK to activate ERM. We also demonstrated that ERM can also be activated through the protein kinase A (PKA), another signaling pathway. Nevertheless, the MAPK and PKA activation of ERM are not synergistic. Finally, we showed that this Ets transcription factor is in vitro phosphorylated by both activated ERK-2 and activated PKA. ERM has thus been identified as a transcription factor which is a target for two different signaling pathways and might therefore be involved in the mitogenic response of cells.