Figure 2.

Activated Raf-1 preferentially interacts with Rok-α. (A–C) EGF increases the Rok-α–Raf-1 interaction. (A) MEFs were stimulated with 10 ng/ml EGF, and endogenous Rok-α was immunoprecipitated at the indicated time points. (B and C) Fluorescence lifetime (τ), GFP intensity, and RFP intensity in MDA-MB-468 transfected with GFP-FL Raf-1 and mRFP–FL Rok-α upon stimulation with 100 ng/ml EGF. (C) Percentage of FRET efficiency is shown. Error bars indicate SEM (n > 3). (D) Activated Ras promotes Rok-α–Raf-1 interaction. COS-1 cells were transfected with HA-tagged FL Rok-α, FL Raf-1, constitutively active Ras (RasV12), or membrane-tethered SOS-F, resulting in the constitutive activation of endogenous Ras and the corresponding vectors (V). UT, untransfected COS-1 cells; *, endogenous Ras. Black lines indicate that intervening lanes have been spliced out. (E–G) Ras binding and subcellular localization affect Rok-α–Raf-1 interaction. (E) COS-1 cells were transfected with HA-tagged FL Rok-α and the indicated FL Raf-1 mutants. HA immunoprecipitates were analyzed and quantified as described in Fig. 1. (F) Fluorescence lifetime, GFP intensity, and mRFP1 intensity in MCF-7 cells transfected with GFP-FL Raf-1 WT or CC/SS mutant (donor) and mRFP1–Rok-α–K (acceptor). The cell marked with the asterisks was excluded from the cumulative FRET efficiency analysis in G as a result of insufficient photon counts (see Materials and methods). (G) Percentage of FRET efficiency is shown. (A, E, and G) Error bars indicate SD of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (B) 20 µm; (F) 30 µm.