Single-molecule analysis of the spatio-temporal properties of cell signaling processes by small GTPases.

一个小的GTPase H-Ras （Ras）是控制细胞内各种信号传导过程的重要分子开关。许多细胞外刺激，如表皮生长因子（EGF）携带的刺激，通过将与Ras结合的鸟嘌呤核苷酸从GDP交换到GTP来激活Ras。GTP结合形式的Ras被细胞质效应蛋白识别，在细胞内转导信号。然而，Ras及其效应物之间相互作用的细节在很大程度上是未知的，因为这种相互作用是弱的和不稳定的。我们已经开发了单分子成像技术，甚至可以在活细胞中进行分子相互作用的动力学分析。在本研究项目中，我们应用该技术研究了Ras与活细胞中Ras效应物之一的c-Raf1 （Raf）相互作用的动力学。结果表明：1)Ras与Raf的相互作用包括解离前的动力学中间体。2) Raf与Ras的两个结合域RBD和CRD都是形成动力学中间体所必需的。3)在初始结合状态下，Raf的RBD和CRD都与Ras结合。因此，CRD与Ras的结合（在RBD结合之后）并不是中间形成的限速步骤。4) Raf在therine/苏氨酸残基中的磷酸化影响中间生成和解离步骤的反应速率。5)在60分钟以上的时间内，Raf在质膜内的积累保持动态平衡。单个Raf分子不断地在质膜和细胞质之间循环。

A small GTPase H-Ras (Ras) is an important molecular switch controlling various intracellular signaling processes. Many extracellular stimulations, such as carried by epidermal growth factor (EGF), activate Ras by exchanging gunanine nucleotide bound to Ras from GDP to GTP. Ras in GTP binding form is recognized by cytoplasmic effecter proteins to transduce signals inside cells. However, details of interactions between Ras and its effectors are largely unknown because the interactions are weak and unstable. We have developed single-molecule imaging technique that allows kinetic analysis of molecular interactions even in living cells. In this research project, we have applied this technique to study kinetics of interaction between Ras and c-Raf1 (Raf) one of the effecters of Ras in living cells.The results can be summarized as follows :1)The interaction between Ras and Raf includes kinetic intermediate before dissociation.2)Both two of the binding domain of Raf to Ras, RBD and CRD are required for the formation of the kinetic intermediate.3)At the initial state of binding, both RBD and CRD of Raf bind to Ras. Thus, binding of CRD to Ras (after binding of RBD) is not the rate limiting step of the intermediate formation.4)Phosphorylations of Raf in therine/threonine residues affect the reaction rate of both intermediate formation and dissociation steps.5)Evident accumulations of Raf in the plasma membrane for more than 60 minutes are maintained as a dynamic equilibrium. Individual molecules of Raf are continuously circulating between the plasma membrane and the cytoplasm.