Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin

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 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.

Figure 1. Photo-initiated cross-linking of the Ca²⁺/CaM with K-RasB-GDP and K-RasB-GppNHp.

Samples containing Ca²⁺/CaM alone (lane 1), Ca²⁺/CaM and K-RasB-GDP (lane 4), or Ca²⁺/CaM and K-RasB-GppNHp (lane 5), were irradiated for 1 minute with a 150-W common flashlight as described in “Materials and Methods”. Samples containing Ca²⁺/CaM and K-RasB-GDP (lane 2), or Ca²⁺/CaM and K-RasB-GppNHp (lane 3) without Ru(bpy)₃Cl₂ and irradiation were also conducted as controls. Shown is a Western blot using antibody raised against CaM.

Figure 2. Interaction of K-RasB with Ca²⁺/CaM at 25.0°C.

Fluorescence spectra (A) of 1.0 µM dansyl-CaM in the absence and in the presence of K-RasB-GppNHp at different concentrations. The arrow represents the concentration of K-RasB-GppNHp increases gradually from 0 (the bottom) to 8.0 µM (the top). ΔF/ΔF _max for the binding of K-RasB-GppNHp to Ca²⁺/CaM plotted as a function of the concentration of K-RasB-GppNHp (B). The solid squares were the experimental data and the solid line represented the best fit. The panel C represents typical calorimetric titration of K-RasB-GppNHp (20.0 µM) with CaM (300 µM) in the presence of 1 mM CaCl₂. The panel D shows the plots of the heat evolved (kcal) per mole of CaM added, corrected for the heat of CaM, against the molar ratio of CaM to K-RasB. The data (solid squares) were fitted to a single set of identical sites model and the solid line represented the best fit. The corresponding parameters from B and D are summarized in Table 1.

Figure 3. Interaction of K-RasB-K175A with Ca²⁺/CaM at 25.0°C.

Fluorescence spectra (A) of 1.0 µM dansyl-CaM in the absence and in the presence of K-RasB-K175A-GppNHp at different concentrations. The arrow represents the concentration of K-RasB-K175A increases gradually from 0 (the bottom) to 14.0 µM (the top). ΔF/ΔF _max for the binding of K-RasB-K175A to Ca²⁺/CaM plotted as a function of the concentration of K-RasB-K175A (B). The solid squares were the experimental data and the solid line represented the best fit. The panel C represents typical calorimetric titration of K-RasB-K175A (35.0 µM) with CaM (800 µM) in the presence of 1 mM CaCl₂. The panel D shows the plots of the heat evolved (kcal) per mole of CaM added, corrected for the heat of CaM, against the molar ratio of CaM to K-RasB-K175A. The data (solid squares) were fitted to a single set of identical sites model and the solid line represented the best fit. The corresponding parameters from B and D are summarized in Table 1.

Figure 4. Interaction of H-Ras, K-RasB-(DESGPC), and H-Ras-(KKKKKK) with Ca²⁺/CaM at 25.0°C.

The panels A, B, and C represent typical calorimetric titration of H-Ras-GppNHp (30.0 µM), K-RasB-(DESGPC)-GppNHp (25.0 µM) and H-Ras-(KKKKKK)-GppNHp (25.0 µM) with CaM (300 µM) respectively in the presence of 1 mM CaCl₂. The panels D, E, and F show the plots of the heat evolved (kcal) per mole of CaM added, corrected for the heat of CaM, against the molar ratio of CaM to Ras proteins. The data (solid squares) were fitted to a single set of identical sites model and the solid line represented the best fit.

Figure 5. Interaction of farnesylated K-RasB with Ca²⁺/CaM at 25.0°C.

Fluorescence spectra (A) of 1.0 µM dansyl-CaM in the absence and in the presence of GppNHp bound K-RasB-farn at different concentrations. The arrow represents the concentration of K-RasB-farn increases gradually from 0 (the bottom) to 3.3 µM (the top). ΔF/ΔF _max for the binding of K-RasB-farn to Ca²⁺/CaM plotted as a function of the concentration of K-RasB-farn (B). The solid squares were the experimental data and the solid line represented the best fit. The panel C represents typical calorimetric titration of K-RasB-farn (30.0 µM) with CaM (600 µM) in the presence of 1 mM CaCl₂. The panel D shows the plots of the heat evolved (kcal) per mole of CaM added, corrected for the heat of CaM, against the molar ratio of CaM to K-RasB. The data (solid squares) were fitted to a single set of identical sites model and the solid line represented the best fit. The corresponding parameters from B and D are summarized in Table 1.

Figure 6. Interaction of farnesylated K-RasB-K175A with Ca²⁺/CaM at 25.0°C.

Fluorescence spectra (A) of 1.0 µM dansyl-CaM in the absence and in the presence of GppNHp bound K-RasB-K175A-farn at different concentrations. The arrow represents the concentration of K-RasB-K175A-farn increases gradually from 0 (the bottom) to 8.4 µM (the top). ΔF/ΔF _max for the binding of K-RasB-K175A-farn to Ca²⁺/CaM plotted as a function of the concentration of K-RasB-K175A-farn (B). The solid squares were the experimental data and the solid line represented the best fit. The panel C represents typical calorimetric titration of K-RasB-K175A-farn (25.0 µM) with CaM (600 µM) in the presence of 1 mM CaCl₂. The panel D shows the plots of the heat evolved (kcal) per mole of CaM added, corrected for the heat of CaM, against the molar ratio of CaM to K-RasB. The data (solid squares) were fitted to a single set of identical sites model and the solid line represented the best fit. The corresponding parameters from B and D are summarized in Table 1.