FRS2 Interaction with Flotillin-1 and CAP Figure 5. Flotillin-1 is required for the recruitment of FRS2 into light membranes in pervanadate treated cells. Hep3B cells (control: upper panels, flot-1 knockdown: lower panels) were starved overnight and then stimulated with pervanadate. Detergent resistant light membranes were prepared using density gradient centrifugation and found in fractions 1–3 of the gradient. The localization of FRS2, flot-1 and CAP was analyzed. Western blots for transferrin receptor (TfnR), GAPDH and GM1-bound cholera toxin subunit B (CTX-B) were used to control the gradient. doi:10.1371/journal.pone.0029739.g005 the FL CAP, whereas the SoHo-GST interacted less strongly (Fig. 6D). Of the three SH3 domains (src-homology domain 3) of CAP, only the third one (SH3C) was found to bind FRS2. Quantification of the binding showed that full-length CAP, DSoHo and SH3C all displayed a significant binding to FRS2 (Fig. 6E). These results imply that multiple domains in FRS2 and CAP are involved in their interaction. These in vitro results were verified by coimmunoprecipitation of the endogenous proteins from Hep3B cells, in which a strong coprecipitation was detected. A similar degree of coprecipitation was detected in control and flot-1 or flot-2 knockdown cells (Fig. 6F), although the coprecipitation may even be slightly increased in flot-1 knockdown cells. Since the SoHo domain of CAP and the PTB domain of FRS2, which were found to interact, are the domains that also mediate the interaction of the respective proteins with flot-1, we tested the hypothesis that CAP and flot-1 might compete for the binding to FRS2. CAP-GST was immobilized on sepharose beads and incubated in the presence of increasing concentra- tions of purified FRS2-His, and the binding of endogenous flot-1 from HeLa lysates was tested (Fig. 7). The presence of increasing concentrations of FRS2-His indeed was capable of abolishing the interaction of flot-1 with CAP, demonstrating that rather than forming a trimeric complex, FRS2, flot-1 and CAP may compete for the binding of each other due to their partly overlapping interaction domains. Since the amount of FRS2 bound by CAP only modestly increases upon incubation with higher amounts of purified FRS2, whereas CAP-bound flot-1 is clearly reduced, FRS2 most likely binds to flot-1 in the lysate, prevents it from binding to CAP and keeps it in the soluble fraction. Discussion Here we have shown that the signaling adaptor protein FRS2 directly interacts both in vivo and in vitro with the membrane raft- associated flot-1. This interaction is mediated by the PTB domain and, to a lesser extent, the C-terminus of FRS2 and by the C- terminus of flot-1. We were able to coprecipitate flot-1 together with FRS2 from mouse tissue lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it [25], does not appear to directly interact with FRS2. However, coprecipitation of flot-2 with FRS2 was detected from cell lysates, which is probably due to the strong association of flot-2 with flot-1 and not a result of a direct interaction with FRS2. Further proof for the functional role of the interaction of flot-1 with FRS2 was provided by our results showing that depletion of flot-1 affects the cellular localization of FRS2 in that it appeared to be more soluble. Furthermore, in vanadate stimulated cells, a small fraction of FRS2 was recruited into detergent resistant membranes, which did not take place in the absence of flot-1. Even more importantly, FRS2 was not able to rescue the phosphorylation deficiency of ERK in flot-1 knockdown cells. Overexpression of FRS2 has previously been shown to result in increased ERK activation [15]. If the ERK activation pathways that flot-1 and FRS2 participate in were separate, one would expect that FRS2 overexpression would result in normal ERK phosphorylation. However, if FRS2 and flot-1 are functionally interconnected, FRS2 probably residing upstream of flot-1, no compensation would take place, as observed here. Thus, the interaction of FRS2 and flot-1 during FGFR signaling may be required for a proper activation of MAP kinases. PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29739