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 透明细胞肉瘤是一种发生在青壮年的侵袭性骨和软组织癌。它是由基因引起的 尤文氏肉瘤癌基因EWS与激活转录因子ATF1或CREB的融合 (cAMP反应元件结合蛋白)，产生由N-末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-末端一半融合。嵌合EWS/atf1和 EWS/CREB癌蛋白是一种有效的转录因子，通过与cAMP反应元件结合。 ATF1和CREB结构域。正常人ATF1和CREB被刺激诱导的磷酸化激活， 其中Ser63处atf1和Ser133处的CREB被蛋白激酶A(PKA)或相关的激酶磷酸化 对各种生长因子和荷尔蒙作出反应。相比之下，分子机制通过这些机制 EWS/atf1和EWS/CREB的转录功能是否受到调控仍不清楚。其主要原因是 这个问题是这样一个事实：融合到EWS的截断的atf1和creb结构域不包含这些规范的 Ser63和Ser133是由Ser63和Ser133介导的PKA磷酸化位点。然而，我们最近确定了保守的新 Atf1在Ser198位和CREB在Ser271位的嵌合癌蛋白的磷酸化位点。有趣的是，我们 发现HIPK2(同源结构域相互作用蛋白激酶2)，而不是PKA，使EWS/atf1在 Ser198，并抑制关键的EWS/atf1靶基因之一c-fos的转录 驱动CCS侵袭性细胞增殖的基因。事实上，EWS/atf1可以在第198位被磷酸化。 人CCS细胞中的ATF1结构域。此外，我们初步确定了atf1ser198的候选者。 磷酸酶。因此，我们发现了两个新的潜在的ews/atf1癌蛋白可逆调节因子。 通过Ser198的磷酸化。我们将检验我们的假设，即EWS/ATF1的磷酸化状态在 Ser198决定EWS/atf1在负责攻击的关键靶基因上的转录活性 CCS癌细胞增殖情况。我们将表征蛋白的磷酸化和去磷酸化。 EWS/atf1基因Ser198位与其转录功能及CCS恶性表型相关。作为atf1和 CREB含有高度保守的磷酸化位点，我们预计与CREB的调节机制相同 截短CREB结构域中Ser271磷酸化的EWS/CREB融合癌蛋白。HIPK2以及 Ser198 atf1磷酸酶可作为选择性更强的新的分子靶点 并在CCS中有效地灭活这些融合癌蛋白。 1