Basis of Phosphorylation in TGF beta signaling

描述(申请者描述)：该项目的长期目标是 理解转化生长因子-β介导的信号转导的结构基础 细胞因子。转化生长因子-β在哺乳动物内分泌中发挥重要而多样的作用 功能。特别是，它们诱导增长的能力阻碍和促进 细胞外基质的形成直接关系到对 糖尿病患者肿瘤增殖失控与肾脏肥大 肾病。I型和II型跨膜受体介导的转化生长因子-β信号 丝氨酸/苏氨酸激酶。配体结合诱导I型-II型受体 结合，导致I型激酶的磷酸化和激活 由II型激酶引起。然后该信号由细胞质Smad3转导， 它是由受体激酶介导的磷酸化激活的。磷酸化 Smad3与Smad4形成异构体络合物，并进入 细胞核来调节转录。这项提议研究的是分子 磷酸化诱导Smad3-Smad4结合的机制。初步 研究表明，SMAD3 C-末端具有直接作用 伪磷酸化在促进Smad3-Smad4相互作用中的作用这个 特定的目标决定了互动的结构基础，使用 生物化学和生物物理方法。定点突变将是 用于作图亚基相互作用面和结合位点 Smad3的磷酸化C-末端尾巴。一种半综合方法，使用 内含素介导的蛋白质连接产生磷酸化形式的Smad3 将被用来解决生理磷酸化对 异构体相互作用。化学交联实验将被用来 探测杂低聚物内的亚单位化学计量比并提供 子单元到子单元的距离信息。沉积速度与平衡 将进行实验以研究关联模型和 异构体缔合的充满活力的。X射线结晶学将被用于 确定详细的分子相互作用。这些方法是相辅相成的 并将现有关于SMAD蛋白质功能的生物信息与 详细的结构性行动基础。阐明这些物质的结构基础 信号相互作用将增加我们对这些过程的理解，并 从而能够产生针对人类疾病的有效治疗试剂。

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.