Basis of Phosphorylation in TGF beta signaling

TGF beta 信号传导磷酸化的基础

• 批准号: 6331546
• 负责人: KAI LIN
• 金额: $ 25.12万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331546
• 关键词: X ray crystallography; biological signal transduction; chromatography; phosphorylation; protein protein interaction; site directed mutagenesis; transcription factor; transforming growth factors

Description (applicant's description): The long-term goal of the project is to understand the structural basis of signaling mediated by the TGF-beta cytokines. TGF-betas play important and diverse roles in mammalian endocrine function. In particular, their ability to induce growth arrest and promote extracellular matrix formation has direct relevance to the understanding of uncontrolled tumor proliferation in cancer and renal hypertrophy in diabetic nephropathy. TGF-betas signal through type I and type II transmembrane receptor serine/threonine kinases. Ligand binding induces type I-type II receptor association, resulting in phosphorylation and activation of the type I kinase by the type II kinase. The signal is then transduced by cytoplasmic smad3, which is activated by receptor kinase-mediated phosphorylation. Phosphorylated smad3 forms a heteromeric complex with smad4, and the complex enters the nucleus to regulate transcription. This proposal examines the molecular mechanism of phosphorylation-induced smad3-smad4 association. Preliminary studies have demonstrated a direct role of smad3 C-terminal pseudophosphorylation in promoting smad3-smad4 interaction in vitro. The specific aims determine the structural basis of the interaction using biochemical and biophysical approaches. Site-directed mutagenesis will be employed to map subunit interacting surface and the binding site for the phosphorylated C-terminal tail of smad3. A semi-synthetic approach using intein-mediated protein ligation to generate the phosphorylated form of smad3 will be used to address the effect of physiological phosphorylation on heteromeric interaction. Chemical cross-linking experiments will be used to probe subunit stoichiometry within the hetero-oligomer and provide subunit-to-subunit distance information. Sedimentation velocity and equilibrium experiments will be performed to investigate the association model and the energetic of heteromeric association. X-ray crystallography will be used to determine the detailed molecular interaction. The approaches are complementary and will link existing biological information on SMAD protein function to the detailed structural basis of action. Elucidating the structural basis for these signaling interactions would increase our understanding of these processes and thereby ability to generate effective therapeutic reagents for human diseases.

描述（申请人的描述）：该项目的长期目标是 了解 TGF-β 介导的信号传导的结构基础 细胞因子。 TGF-β 在哺乳动物内分泌中发挥着重要而多样的作用 功能。特别是，它们诱导生长停滞和促进生长的能力 细胞外基质的形成与理解有直接关系 癌症中不受控制的肿瘤增殖和糖尿病中的肾肥大 肾病。 TGF-β 通过 I 型和 II 型跨膜受体发出信号 丝氨酸/苏氨酸激酶。配体结合诱导I型-II型受体 关联，导致 I 型激酶磷酸化和激活 由 II 型激酶。然后信号由细胞质 smad3 转导， 它由受体激酶介导的磷酸化激活。磷酸化 smad3与smad4形成异聚复合物，该复合物进入 细胞核来调节转录。该提案检查了分子 磷酸化诱导的 smad3-smad4 关联机制。初步的 研究表明 smad3 C 末端的直接作用 假磷酸化在体外促进 smad3-smad4 相互作用。这 具体目标决定了交互的结构基础 生物化学和生物物理方法。定点诱变将 用于绘制亚基相互作用表面和结合位点 smad3 磷酸化的 C 末端尾部。半合成方法使用 内含肽介导的蛋白质连接产生磷酸化形式的 smad3 将用于解决生理磷酸化对 异聚相互作用。化学交联实验将用于 异源低聚物内的探针亚基化学计量并提供 子单元到子单元的距离信息。沉降速度和平衡 将进行实验来研究关联模型和 充满活力的异聚体关联。 X射线晶体学将用于 确定详细的分子相互作用。这些方法是互补的 并将 SMAD 蛋白功能的现有生物学信息与 行动的详细结构基础。阐明这些的结构基础 信号相互作用将增加我们对这些过程的理解 从而能够产生针对人类疾病的有效治疗试剂。