Figure 5.

MCP110 impairs recruitment of Raf-RBD to subcellular locations of active Ras. A. Recruitment of YFP-Raf-RBD to sites where Ras is localized, as detected by fluorescence microscopy. NIH3T3 cells transiently expressing both HA-tagged active Ras and YFP-Raf-RBD were treated with vehicle or MCP110. Shown are representative images of cells quantitated in Panel B below. In the absence of active Ras (v.o.), YFP-Raf-RBD (green) was localized diffusely throughout the cytoplasm and nucleus, whereas in the presence of active Ras, the Raf-RBD probe was recruited to the plasma membrane and, like Ras (red), was nuclear-excluded ("vehicle" panels). Increasing concentrations of MCP110 increasingly shifted the YFP-Raf-RBD probe from the plasma membrane to the cytosol and to internal membranes and finally to both cytosol and nucleus, whereas Ras remained membrane-associated and nuclear-excluded. These results indicate dose-dependent disruption of the Ras/Raf-RBD interaction by MCP110. B. Quantification of the distribution of YFP-Raf-RBD subcellular localization. Cells treated and analyzed as described in Methods and depicted qualitatively in Panel C were binned according to whether the YFP-Raf-RBD probe accumulated primarily in the cytosol, or cytosol + internal membranes, or at the plasma membrane and was nuclear-excluded. MCP110 dose-dependently disrupts the ability of Ras to recruit Raf-RBD.