INTRACELLULAR SIGNALING PHENOTYPE FOR THYMOCYTE

胸腺细胞的细胞内信号传导表型

• 批准号: 2004448
• 负责人: Christopher E. Rudd
• 金额: $ 16.32万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2004448
• 关键词: T lymphocyte; binding proteins; biological signal transduction; cell differentiation; interleukin 2; laboratory mouse; phenotype; phosphorylation; protein biosynthesis; protein kinase; thymus; tissue /cell culture; transfection

DESCRIPTION (Adapted from the Applicant's Abstract): Thymic differentiation involves a discrete series of stages leading to positive and negative selection. Much is known regarding changes in the cell surface phenotype during T cell maturation. By contrast, little is understood regarding the intracellular signaling mechanisms that control differentiation. The applicant has previously reported the identification of a 120/130 kDa protein that binds to the SH2 domain of the src-family tyrosine kinase Fyn. The applicant believes that these polypeptides correspond to CAS, an adapter protein with multiple YDXP motifs and a single SH3 domain. These pYDXP motifs are ideally suited to bind to the SH2 domain of the intracellular protein CRK. Recently, the applicant has found that thymocytes exhibit a highly restricted pattern of tyrosine phosphorylation with only two major sets of phosphorylated substrates at 120/130kDa and 50-65kDa, in contrast to mature T cells, where the pattern is much more complex. Anti-120/130 antibodies immunoprecipitated the majority of the prominently tyrosine phosphorylated polypeptides from thymocytes. This suggests that CAS and associated proteins make up the majority of tyrosine phosphorylated proteins in thymocytes. This observation suggests that CAS and associated proteins may play important roles in thymic differentiation. In this application, the investigator proposes to study further the role of CAS and associated proteins in signaling in thymocytes and mature T cells by exploring the interaction between CAS, CRK, and the guanine nucleotide releasing factor C3G. C3G may be involved in the conversion of Ras to a GTP-bound active state, an event that is central to the control of cellular growth and differentiation. In Aim 2, the applicant will attempt to isolate a cDNA encoding a T-cell specific form of CAS. An antisera generated against p120/130 isolated from T cells is restricted in its recognition of the T cell form, termed CAS-T. In Aim 3, the applicant will transfect a T cell hybridoma DC.27 with forms of CAS or CAS-T for analysis of its role in production of IL-2. Lastly, in Aim 4, the applicant will generate CAS or CAS-T knockout mice for analysis of the role of CAS in thymic development.

描述（改编自申请人的摘要）：胸腺分化 涉及一系列离散的阶段，导致积极和消极 选择。 关于细胞表面表型的变化已知很多 T细胞成熟期间。 相比之下，人们对 控制分化的细胞内信号传导机制。 这 申请人之前已报告过 120/130 kDa 的鉴定 与 src 家族酪氨酸激酶 Fyn 的 SH2 结构域结合的蛋白质。 申请人认为这些多肽对应于CAS，一种衔接子 具有多个 YDXP 基序和单个 SH3 结构域的蛋白质。 这些 pYDXP 基序非常适合结合细胞内的 SH2 结构域 蛋白质CRK。 最近，申请人发现胸腺细胞表现出 酪氨酸磷酸化的高度受限模式只有两个主要 120/130kDa 和 50-65kDa 的磷酸化底物组，与 成熟的 T 细胞，其模式要复杂得多。 抗120/130 抗体免疫沉淀大部分突出的酪氨酸 来自胸腺细胞的磷酸化多肽。 这表明 CAS 和 相关蛋白构成酪氨酸磷酸化蛋白的大部分 在胸腺细胞中。 这一观察结果表明 CAS 和相关蛋白 可能在胸腺分化中发挥重要作用。 在这个应用程序中， 研究者建议进一步研究 CAS 和相关的作用 通过探索胸腺细胞和成熟 T 细胞信号传导中的蛋白质 CAS、CRK 和鸟嘌呤核苷酸释放因子之间的相互作用 C3G。 C3G 可能参与 Ras 向 GTP 结合活性物质的转化 状态，对于细胞生长和控制至关重要的事件 差异化。 在目标 2 中，申请人将尝试分离 cDNA 编码 T 细胞特定形式的 CAS。 产生的抗血清 从 T 细胞中分离出的 p120/130 在识别 T 细胞方面受到限制 细胞形式，称为 CAS-T。 在目标 3 中，申请人将转染 T 细胞 具有 CAS 或 CAS-T 形式的杂交瘤 DC.27，用于分析其在 IL-2的产生。 最后，在目标 4 中，申请人将生成 CAS 或 CAS-T 敲除小鼠用于分析 CAS 在胸腺发育中的作用。