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

小分子GTP酶H-RAS(RAS)是控制细胞内各种信号转导过程的重要分子开关。许多细胞外刺激，如由表皮生长因子(EGF)携带的，通过将与RAS结合的鸟氨酸核苷酸从GDP交换为GTP来激活RAS。Ras以GTP结合的形式被细胞质效应蛋白识别，在细胞内传递信号。然而，RAS与其效应器之间的相互作用的细节在很大程度上是未知的，因为这种相互作用很弱且不稳定。我们已经开发了单分子成像技术，即使在活细胞中也可以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RAS的效应物之一c-Raf1(RAF)在活细胞中的相互作用动力学。结果如下：1)RAF与RAF的相互作用在解离前包括动力学中间体；2)RAF与RAS的结合域都需要RBD和CRD两个结合域来形成动力学中间体。3)在结合的初始状态，RAF的RBD和CRD都与RAS结合。因此，CRD与RAS的结合(在RBD结合后)不是中间体形成的速度限制步骤。4)茶氨酸/苏氨酸残基中Raf的磷酸化影响中间体形成和解离步骤的反应速度。5)Raf在质膜中明显积累超过60min保持动态平衡。Raf的单个分子在质膜和细胞质之间持续循环。

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.