FRS2 Interaction with Flotillin-1 and CAP Figure 2. Endogenous flot-1 and flot-2 colocalize with FRS2 in Hep3B cells. Cells were grown in a medium containing FCS and stained with antibodies against endogenous FRS2 (green) and flot-1 or flot-2 (red). Scale bars 10 mm. doi:10.1371/journal.pone.0029739.g002 control and flot-1 knockdown cells. Thus, although the growth factor induced phosphorylation of FRS2 appears to take place in flot-1 knockdown cells, FRS2 is not able to compensate for the observed downstream signaling defects. FRS2 binds to FGFR cytoplasmic domain by means of its PTB domain but independently of Tyr phosphorylation [6]. Since we could show that this domain also at least partly mediates the interaction to flot-1, we tested if FGFR and flot-1 would compete for the binding to FRS2 (Fig. 3B). For this, we used purified FRS2- GST immobilized on beads and lysates of Hela cells transfected with increasing amounts of myc-tagged FGFR (0.5–2 mg of DNA used for transfection, Fig. 3D). As a transfection control, the empty vector pcDNA3 was used. The binding of endogenous flot-1 to FRS2-GST in the presence of FGFR was found to decrease the more FGFR was present in the lysates (Fig. 3B), but we were unable to obtain a full loss of flot-1 binding by FRS2 due to FGFR. Significantly reduced binding of flot-1 to FRS2 was observed upon increased expression of FGFR (Fig. 3C). For studies of the function of endogenous FRS2, which is expressed in HeLa cells only in very minor amounts, in the absence of flot-1, we generated stable Hep3B clones in which flot-2 or flot-1 were knocked-down using lentivirus-mediated RNA interference. It has to be noted that knockdown of flot-2 results in a concomitant destabilization and downregulation of flot-1 at the protein level, whereas flot-1 depletion only mildly affects flot-2 expression (Suppl. Fig. S1C), as shown by us and others for various cell lines [25,36]. Suppl. Fig. S1D shows that very little flot-2 or flot-1 could be detected by means of immunofluorescence staining in the knockdown cells, demonstrating that a high degree of depletion of flotillins was achieved in virtually all cells of the respective clone. To see if knockdown of flotillins affects the phosphorylation status of FRS2, we used the stable Hep3B knockdown cells. FRS2 was immunoprecipitated from serum-grown cells, and the blots were probed with anti-phospho-Tyr antibodies (Fig. 4A). Interest- ingly, we could detect a tendency to increased Tyr phosphoryla- tion of FRS2 in flot-2 knockdown cells and a significantly increased phosphorylation in the flot-1 knockdown cells (Fig. 4B). Since Tyr and Thr phosphorylation of FRS2 have been described to be reciprocally regulated [14,15], one would expect that the increased Tyr phosphorylation would result in decreased Thr phosphorylation of FRS2 in flotillin knockdown cells. We thus aimed at detecting the Thr phosphorylation of FRS2, which is mediated by ERK, by means of antibodies directed against phosphorylated recognition sites of the ERK kinase. Unfortunate- ly, although a tendency to decreased Thr phosphorylation of FRS2 was indeed visible after depletion of flot-2 or flot-1, the signals were generally very weak and did not allow for any definite conclusions (Data not shown). FRS2 has been described to associate with cellular membranes by means of myristoylation. Since myristoylation alone can only provide a weak membrane attachment [37], we studied by means of immunofluorescence studies if the cellular localization of FRS2 might depend on flotillins. Although FRS2 was localized at the plasma membrane in the control cells, knockdown of flot-1 or flot- 2 resulted in a slight increase in the cytoplasmic staining and some loss of membrane localization of FRS2 (Fig. 4C), indicating that in the absence of flotillins, FRS2 is less membrane associated. In addition, some nuclear staining for FRS2 was detected in flot knockdown cells but not in the control. These results suggest that the membrane association of FRS2 may be facilitated by the presence of flotillins. Since flot-2 and flot-1 knockdown cells gave virtually identical results in these assays, it is likely that the changes in FRS2 membrane association are more attributable to the absence of flot-1 expression, considering that flot-2 knockdown cells also lack flot-1 (Suppl. Fig. S1B). Previous data have shown that in some cell types, FRS2 is constitutively associated with membrane rafts [38,39]. Thus, the interaction between flot-1 and FRS2 might facilitate the localization of FRS2 into these domains. To test this, we again used the Hep3B cells which were either serum starved or starved and then stimulated with pervanadate to enhance Tyr phosphor- ylation. Detergent resistant membranes (referred to as rafts below) were isolated using density gradients after detergent extraction [40]. In these gradients, rafts are mainly present in fractions 1–3, as evidenced by the enrichment of cholera toxin in these fractions PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29739