both the c-terminal polylysine region and the farnesylation of k-rasb are important for its specific interaction with calmodulin的c端赖氨酸地区和farnesylation k-rasb与钙调蛋白为其特定的互动很重要.pdfVIP

Both the C-Terminal Polylysine Region and the Farnesylation of K-RasB Are Important for Its Specific Interaction with Calmodulin Ling-Jia Wu, Li-Rong Xu, Jun-Ming Liao, Jie Chen, Yi Liang* State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China Abstract Background: Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood. Methodology/Principal Findings: Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca2+/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K- RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca2+/CaM formed a 1:1 complex with an equilibrium association constant around 105 M21, whereas no binding reaction of K-RasB- (DESGPC) with Ca2+/CaM is detected. Furthermore, the interaction of K-RasB with Ca2+/CaM is found to be enhanced by the farnesylation of K-RasB. Conclusions/Significance: We demonstrate that the polylysine region of K-RasB not only contributes i