AATYK--A NOVEL APOPTOSIS ASSOCIATED TYROSINE KINASE

AATYK--一种新型凋亡相关酪氨酸激酶

• 批准号: 6329463
• 负责人: E Premkumar Reddy
• 金额: $ 21.22万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329463
• 关键词: animal genetic material tag; antisense nucleic acid; apoptosis; cell differentiation; developmental genetics; enzyme activity; enzyme induction /repression; enzyme inhibitors; etoposide; genetic regulatory element; granulocyte; immunogenetics; methotrexate; mitomycin C; posttranslational modifications; protein tyrosine kinase; tissue /cell culture

DESCRIPTION: (Adapted from the Applicant's Abstract) To study the nature of genes that are induced during the apoptotic death of myeloid pre-cursor cells, the investigators utilized 32Dcl3 cell line, which is derived from normal mouse bone marrow and is non-tumorigenic and diploid. These cells are strictly dependent on IL-3 for growth and apoptosis when deprived of IL-3 from the medium. In the search for genes that are induced during terminal differentiation of 32Dcl3 cells, the investigators identified a novel gene termed AATYK (Apoptosis Associated Tyrosine Kinase), whose expression is dramatically upregulated during IL-3 deprivation. The expression of this gene, which codes for a protein with a tyrosine kinase domain at the N-terminal end and a proline-rich domain at the C-terminal end, is blocked in transformed myeloid cells which are deficient in undergoing apoptosis. The experiments proposed are aimed at understanding the role of AATYK in the apoptosis, differentiation, and transformation of myeloid cells. The aims are: 1) [a] to test whether other apoptotic stimuli produced by different xenobiotic agents such as calphostin C, methotrexate etoposide, and mitomycin C result in the induction of AATYK, [b] to test whether ectopic over-expression of AATYK renders v-abl and bcr-abl-transformed 32D cells more sensitive to apoptotic death induced by the above xenobiotic agents, and [c] to test whether transgenic expression of AATYK in the v-abl or bcr-abl transformed 32D cells over-rides the block to G-CSF-induced terminal differentiation; 2) to study the effects of transgenic expression of AATYK on 32Dcl3 cell growth, differentiation, and apoptosis and to determine as to how myeloid cell differentiation (in the presence of GCSF) or apoptosis (in the absence of IL-3) is affected when AATYK expression is inhibited using anti-sense vectors; 3) to carry out a detailed biochemical characterization of the protein encoded by AATYK to determine its potential tyrosine kinase activity, post-translational modification patterns, subcellular localization and mechanism of action; and, 4) to carry out a detailed analysis of the promoter/enhancer region of AATYK to examine the sequence elements that play a crucial role in the transcriptional regulation of this gene.

描述：（改编自申请人的摘要）为了研究 在骨髓前体细胞凋亡过程中诱导的基因 细胞，研究人员利用32 Dc 13细胞系，其来源于 正常小鼠骨髓，并且是非致瘤性和二倍体。 这些细胞 在缺乏IL-3时，它们的生长和凋亡严格依赖于IL-3。 培养基中的IL-3。 在寻找基因的过程中， 在32 Dcl 3细胞的终末分化中，研究人员发现了一种 一种新的基因AATYK（凋亡相关酪氨酸激酶）， 表达在IL-3剥夺期间显著上调。 的 该基因编码具有酪氨酸激酶的蛋白质， 在N-末端的结构域和在C-末端的富含脯氨酸的结构域 终末，在转化的髓样细胞中被阻断， 正在经历凋亡。 所提出的实验旨在了解 AATYK在细胞凋亡、分化和转化中的作用 骨髓细胞 目的是：1）[a]测试其他凋亡 由不同的异生素试剂如钙磷蛋白C产生的刺激， 甲氨蝶呤、依托泊苷和丝裂霉素C导致AATYK的诱导， [b]为了测试AATYK的异位过表达是否使v-abl和 bcr-abl-转化的32 D细胞对诱导的凋亡更敏感 上述异生素试剂，和[c]测试转基因表达是否 在v-abl或bcr-abl转换的32 D单元中的AATYK覆盖块 G-CSF诱导的终末分化; 2）研究G-CSF对终末分化的影响， AATYK的转基因表达对32 Dcl 3细胞生长、分化和 细胞凋亡，并确定如何髓样细胞分化（在 GCSF的存在）或细胞凋亡（不存在IL-3）受到影响， 使用反义载体抑制AATYK表达; 3）进行反义寡核苷酸表达。 AATYK编码的蛋白质的详细生物化学表征， 确定其潜在的酪氨酸激酶活性，翻译后 修饰模式、亚细胞定位和作用机制; 以及4）对以下的启动子/增强子区域进行详细分析： AATYK来检查序列元件，这些序列元件在基因组中起着至关重要的作用。 该基因的转录调控。