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

• 批准号: 10513111
• 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
• 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/10513111
• 关键词: Antineoplastic Agents; Automobile Driving; Binding; Bone Tissue; C-terminal; 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; Protein Binding Domain; Protein Dephosphorylation; Protein Kinase; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Role; Site; Somatotropin; Stimulus; Survival Rate; Tertiary Protein Structure; Testing; Tumor Suppressor Proteins; activating transcription factor; anticancer research; bZIP Domain; cancer cell; cell growth; experimental study; extracellular; homeodomain; malignant phenotype; novel; programs; response; soft tissue; transcription factor; young adult

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

摘要 透明细胞肉瘤（CCS）是一种侵袭性骨和软组织癌症的年轻成年人。这是由基因引起的 EWS（尤因肉瘤癌基因）和ATF 1（转录激活因子）或CREB之间的融合 （cAMP-反应元件结合蛋白），产生由N-末端 EWS结构域融合到截短的ATF 1和CREB结构域的C-末端一半。嵌合EWS/ATF 1和 EWS/CREB癌蛋白是有效的转录因子，其通过EWS/CREB结合CRE（cAMP反应元件）。 ATF 1和CREB结构域。正常人ATF 1和CREB通过刺激诱导的磷酸化激活， 其中位于Ser 63的ATF 1和位于Ser 133的CREB被蛋白激酶A（PKA）或相关激酶磷酸化 对各种生长因子和激素的反应。相反，通过分子机制， EWS/ATF 1和EWS/CREB的转录调控功能尚不清楚。的主要原因 这个问题是与EWS融合的截短的ATF 1和CREB结构域不包含这些典型的 Ser 63和Ser 133 PKA介导的磷酸化位点。然而，我们最近发现了保守的新的 在嵌合癌蛋白ATF 1的Ser 198和CREB的Ser 271的磷酸化位点。有趣的是，我们 发现HIPK 2（同源结构域相互作用蛋白激酶2），而不是PKA，磷酸化EWS/ATF 1， ATF 1结构域的Ser 198并抑制c-FOS的转录，c-FOS是EWS/ATF 1的关键靶点之一。 基因驱动CCS的细胞增殖。事实上，EWS/ATF 1可以在蛋白质的Ser 198处磷酸化， 人CCS细胞中的ATF 1结构域。此外，我们初步鉴定了ATF 1 Ser 198的候选者， 磷酸酶因此，我们发现了两个新的潜在的EWS/ATF 1癌蛋白可逆调节剂， Ser 198磷酸化我们将检验我们的假设，即EWS/ATF 1的磷酸化状态在 Ser 198决定了EWS/ATF 1在负责侵袭性肿瘤的关键靶基因上的转录活性。 CCS癌细胞的增殖。我们将描述的磷酸化和去磷酸化的 EWS/ATF 1的Ser 198位点与其转录功能和CCS恶性表型相关。ATF 1和 CREB含有高度保守的磷酸化位点，我们预期CREB的调节机制与CREB的磷酸化位点相同。 EWS/CREB融合癌蛋白通过截短的CREB结构域中的Ser 271磷酸化。HIPK 2以及 Ser 198 ATF 1磷酸酶可以作为一种新的分子靶点， 以及CCS中这些融合癌蛋白的有效失活。 1