AATYK--A NOVEL APOPTOSIS ASSOCIATED TYROSINE KINASE

描述：(改编自申请人的摘要)研究 髓系前体细胞凋亡死亡过程中诱导的基因 细胞，研究人员利用了32Dcl3细胞系，它来自于 正常小鼠骨髓，无致瘤性，为二倍体。这些细胞 在被剥夺时严格依赖IL-3的生长和凋亡 IL-3从培养基中分离出来。在寻找基因的过程中 在32Dcl3细胞的终末分化中，研究人员发现了一种 新基因AATYK(凋亡相关酪氨酸激酶)，其 在IL-3剥夺过程中，表达显著上调。这个 该基因的表达，它编码一种带有酪氨酸激酶的蛋白质 N-末端的结构域和C-末端的富含脯氨酸的结构域 末端，在转化的髓系细胞中被阻断，这些细胞缺乏 正在经历细胞凋亡。提出的实验目的是为了了解 AATYK在细胞凋亡、分化和转化中的作用 髓系细胞。其目的是：1)[a]测试其他细胞凋亡 由不同的异源物质产生的刺激，如钙磷蛋白C， 甲氨蝶呤、依托泊苷和丝裂霉素C可诱导AATYK， [B]检测AATYK的异位过表达是否导致v-ABL和 BCR-ABL转化的32D细胞对AFP诱导的细胞死亡更敏感 上述异源药物，以及[c]检测转基因是否表达 在v-abl或bcr-abl转换的32D单元中的AATYK的覆盖块 对G-CSF诱导的终末分化的影响；2)研究 AATYK基因在32Dcl3细胞生长、分化和分化中的转基因表达 并确定髓系细胞如何分化(在 GCSF的存在)或细胞凋亡(在没有IL-3的情况下)在以下情况下受到影响 使用反义载体抑制AATYK的表达；3)进行 AATYK TO编码蛋白的详细生化特性 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)对其启动子/增强子区域进行详细分析 AATYK研究在基因序列中起关键作用的序列元件 该基因的转录调控。

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.