Both the C-Terminal Polylysine Region and the Farnesylation of K-RasB Are Important for Its Specific Interaction with Calmodulin

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 Ca(2+)/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 Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/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 importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.