Elucidation of New Phosphorylation Site of the EWS/ATF1 Fusion Oncoprotein in Clear Cell Sarcoma

透明细胞肉瘤中 EWS/ATF1 融合癌蛋白新磷酸化位点的阐明

基本信息

批准号：
10670347
负责人：
YOSHIAKI TSUJI
金额：
$ 7.6万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10670347
关键词：
Antineoplastic Agents Automobile Driving Binding Bone Tissue C-terminal Cancer Research Project Cell Proliferation Cells Chemical Agents Chemicals Childhood Chimeric Proteins Chromosomal translocation Clear Cell Sarcoma Conserved Sequence Cues Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic AMP-Responsive DNA-Binding Protein DNA Damage Elements Ewings sarcoma FOS gene Fusion Oncogene Proteins Gene Expression Gene Fusion Genes Genetic Transcription Genotoxic Stress Grant Growth Factor Human Investigation Malignant Neoplasms Mediating Molecular Molecular Target N-terminal Nuclear Nuclear Protein Oncogenes Oncogenic Oncoproteins Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Pilot Projects Play Proliferating Protein Binding Domain Protein Dephosphorylation Protein Kinase Protein Kinase Interaction Protein phosphatase Protein-Serine-Threonine Kinases Proteins Role Serine Site Somatotropin Stimulus Survival Rate Testing Tumor Suppressor Proteins activating transcription factor bZIP Domain cancer cell candidate identification cell growth experimental study extracellular homeodomain malignant phenotype novel response soft tissue transcription factor young adult

项目摘要

Abstract Clear cell sarcoma (CCS) is an aggressive bone and soft tissue cancer of young adults. It is caused by gene fusions between EWS (Ewing’s sarcoma oncogene) and ATF1 (activating transcription factor) or CREB (cAMP-responsive element binding protein), giving rise to chimeric oncoproteins consisting of the N-terminal EWS domain fused to a C-terminal half of truncated ATF1 and CREB domains. The chimeric EWS/ATF1 and EWS/CREB oncoproteins are potent transcription factors that bind to CRE (cAMP-responsive element) via the ATF1 and CREB domains. Normal human ATF1 and CREB are activated by stimulus-induced phosphorylation, in which ATF1 at Ser63 and CREB at Ser133 are phosphorylated by protein kinase A (PKA) or related kinases in response to various growth factors and hormones. In contrast, the molecular mechanism through which the transcription function of EWS/ATF1 and EWS/CREB is regulated remains unknown. The primary reason for this problem is the fact that truncated ATF1 and CREB domains fused to EWS do not contain these canonical Ser63 and Ser133 PKA-mediated phosphorylation sites. However, we recently identified the conserved new phosphorylation sites in the chimeric oncoproteins of ATF1 at Ser198 and CREB at Ser271. Intriguingly, we found that HIPK2 (homeodomain interacting protein kinase 2), but not PKA, phosphorylates EWS/ATF1 at Ser198 of the ATF1 domain and suppresses the transcription of c-FOS, one of the key EWS/ATF1 target genes driving aggressive cell proliferation of CCS. Indeed, EWS/ATF1 can be phosphorylated at Ser198 of the ATF1 domain in human CCS cells. Furthermore, we preliminary identified candidates of ATF1 Ser198 phosphatases. Thus, we have found two new potential reversible regulators of EWS/ATF1 oncoprotein through Ser198 phosphorylation. We will test our hypothesis that the phosphorylation status of EWS/ATF1 at Ser198 determines the EWS/ATF1 transcription activity on key target genes responsible for aggressive proliferation of CCS cancer cells. We will characterize the phosphorylation and dephosphorylation of EWS/ATF1 at Ser198 associated with its transcription function and CCS malignant phenotype. As ATF1 and CREB contain highly conserved phosphorylation sites, we anticipate the same regulatory mechanism of the EWS/CREB fusion oncoprotein via Ser271 phosphorylation in the truncated CREB domain. HIPK2 as well as Ser198 ATF1 phosphatases characterized in this proposal can be a new molecular target for more selective and effective inactivation of these fusion oncoproteins in CCS. 1

抽象的透明细胞肉瘤（CCS）是一种侵袭性的年轻人骨和软组织癌症，由基因引起。 EWS（尤文氏肉瘤癌基因）和 ATF1（激活转录因子）或 CREB 之间的融合（cAMP 反应元件结合蛋白），产生由 N 端组成的嵌合癌蛋白 EWS 结构域与截短的 ATF1 和 CREB 结构域的 C 端一半融合嵌合 EWS/ATF1 和 EWS/CREB 癌蛋白是有效的转录因子，通过 ATF1 和 CREB 结构域被刺激诱导的磷酸化激活。其中 Ser63 处的 ATF1 和 Ser133 处的 CREB 被蛋白激酶 A (PKA) 或相关激酶磷酸化相反，通过其分子机制来响应各种生长因子和激素。 EWS/ATF1 和 EWS/CREB 的转录功能受到调节的主要原因仍不清楚。这个问题是这样的事实：与 EWS 融合的截断 ATF1 和 CREB 域不包含这些规范然而，我们最近发现了保守的新磷酸化位点。有趣的是，我们发现了 ATF1 Ser198 和 CREB Ser271 嵌合癌蛋白的磷酸化位点。发现 HIPK2（同源结构域相互作用蛋白激酶 2）而非 PKA 在以下位置磷酸化 EWS/ATF1： ATF1 结构域的 Ser198 并抑制 c-FOS（EWS/ATF1 关键靶标之一）的转录事实上，EWS/ATF1 的 Ser198 可以被磷酸化。此外，我们初步鉴定了人类 CCS 细胞中的 ATF1 结构域 Ser198。因此，我们发现了 EWS/ATF1 癌蛋白的两种新的潜在可逆调节因子。我们将通过 Ser198 磷酸化来检验我们的假设，即 EWS/ATF1 的磷酸化状态为 Ser198 决定了负责攻击性的关键靶基因上的 EWS/ATF1 转录活性我们将表征 CCS 癌细胞的磷酸化和去磷酸化。 Ser198 处的 EWS/ATF1 与其转录功能和 CCS 恶性表型相关。 CREB含有高度保守的磷酸化位点，我们预计与CREB具有相同的调节机制 EWS/CREB 融合癌蛋白通过截短的 CREB 结构域中的 Ser271 磷酸化。本提案中表征的 Ser198 ATF1 磷酸酶可以成为更具选择性的新分子靶标以及 CCS 中这些融合癌蛋白的有效失活。 1