FRS2 Interaction with Flotillin-1 and CAP Figure 3. Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells. FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 mg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot. doi:10.1371/journal.pone.0029739.g003 (Fig. 5). Flot-1 was found to reside in fractions 2–5 in starved cells, whereas in pervanadate stimulated cells, a shift towards raft fractions was observed (Fig. 5, upper part). We were not able to detect FRS2 in the raft fractions in starved cells, whereas in pervanadate stimulated cells, some FRS2 was found in fractions 2– 3 together with flot-1. Intriguingly, pervanadate did not induce a shift of FRS2 towards raft fractions in flot-1 knockdown cells (Fig. 5, lower part), implicating that flot-1 is necessary for the stimulation induced recruitment of FRS2 into detergent insoluble membranes in these cells. However, only a fraction of FRS2 becomes raft associated during signaling, contrary to previous findings from other cell types [38,39]. Flot-1 has previously been described to interact with Cbl- associated protein (CAP). We thus tested in our raft fractions if the localization of CAP would be altered after flot-1 knockdown. In control cells, CAP displayed a broad distribution in the heavier fractions, without localizing to rafts. Although CAP underwent a shift towards lighter membranes upon pervanadate stimulation, it remained concentrated in fractions 5 and 6, without localizing to rafts (Fig. 5, upper part). In flot-1 knockdown cells, CAP was found to reside in fractions 5 and 6 both in starved and stimulated cells (Fig. 5, lower part). Thus, although CAP exhibits a flot-1 dependent localization in these gradients, flot-1 does not recruit CAP into rafts. Previous findings have shown that during signaling of the neurotrophic receptor TrkA, flot-1, FRS2 and CAP all localized into rafts [38]. However, a direct molecular connection between these three proteins has so far not been established. Since flot-1 interacts with both FRS2 and CAP, we tested if FRS2 would be capable of interacting with CAP. Our Y2H analysis showed that FL CAP indeed interacted with FL, PTB and CT domain of FRS2, whereas the sorbin homology (SoHo) domain of CAP only showed an interaction with FRS2 PTB domain (Fig. 6A). Since both FRS2 and CAP posses multiple domains that mediate protein-protein interactions and both CT and PTB in FRS2 appeared to interact with CAP, we used GST pulldown to characterize the interaction domains more closely. Various GST fusion proteins containing CAP domains were generated (Fig. 6B). The purified GST proteins were incubated with purified FRS2-His. A strong interaction was seen between FL proteins (Fig. 6C and D). Interestingly, a CAP mutant lacking the SoHo domain (DSoHo) showed a similar binding to FRS2 as PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29739