Figure 7. Flot-1 and CAP compete for the binding to FRS2. CAP- GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 mg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins. doi:10.1371/journal.pone.0029739.g007 Both the PTB domain and CT of FRS2 were found to mediate this interaction, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. In our GST pulldown assays, deletion of the CAP SoHo domain did not result in a major reduction in binding to FRS2. Furthermore, both the FL CAP and the third SH3 domain showed about similar binding efficiency to FL FRS2, implicating a co-operative mode of binding. SH3 domains bind to proline rich sequences, several of which are present in FRS2, one of them even residing within the PTB sequence [2]. In the Y2H assay, the SoHo domain only bound to the PTB domain, whereas FL CAP bound to FRS2 CT as well. Thus, this would suggest that the SH3 domain of CAP may also be capable of interacting with the poly-Pro sequences in the more C- terminal part of FRS2. Interestingly, CAP has previously been linked to flot-1 during insulin signaling [26]. Furthermore, it has been postulated that the activated NGF receptor TrkA is translocated into flotillin- containing membrane rafts. Differentiation and neurite growth of PC12 cells can be induced by NGF, which binds to its receptor TrkA and induces a signal transduction through rafts. In PC12 cells, a very transient association of TrkA with CAP, which in turn interacted with flot-1 with similar kinetics, was observed. However, CAP was detected in a constitutive complex with an adaptor protein APS [43], which has been shown to associate only with the activated TrkA [44]. This would suggest that during TrkA signaling, CAP associates with flot-1 and activated TrkA only very shortly, possibly mediating raft recruitment of TrkA, but then dissociates from them. Intriguingly, FRS2 has been shown to reside in the rafts in which the activated TrkA is recruited [38], and it is already long recognized that FRS2 is an important regulator of TrkA signaling [2,3,11,14,17,38,45], and may even FRS2 Interaction with Flotillin-1 and CAP participate in insulin signaling [18]. However, FRS2 has never been connected either with flot-1 or CAP before. Since flotillins were originally discovered as neuronal regeneration proteins [32], and knockdown of flotillins in neuronal N2A cells causes reduced neurite outgrowth [31], flotillins might indeed modulate neuronal differentiation together with FRS2 and CAP, most likely by regulating the activation of MAP kinases. We here show that FRS2 can bind to both flot-1 and CAP, whereas previous studies have shown that CAP and flot-1 also interact with each other, and this interaction also requires the SoHo domain of CAP [26,33]. The interaction of flot-1 with FRS2 may be required for the recruitment FRS2 to membranes or even membrane rafts, as has been suggested for the interaction of flot-1 with CAP [33]. This might in turn be necessary for a productive interaction of FRS2 with its signaling partners, such as FGF receptor. Although we cannot exclude that a trimeric complex between FRS2, flot-1 and CAP exists in the cells, the overlapping binding domains would rather suggest that these proteins compete for the binding to each other, as we also observed for the binding of FGFR and flot-1 to FRS2. Alternatively, FRS2, CAP and flot-1 may bind to each other in a successive manner, thus regulating the formation of downstream signaling complexes. In line with this, FRS2 has been shown to be connected by means of the growth factor binding protein-2 (Grb2) with the ubiquitin ligase cCbl during FGFR signaling [46]. On the other hand, cCbl regulates insulin receptor signaling by means of its interaction with CAP, which then binds to flot-1 [26]. Thus, the signalosomes that are formed by FRS2, flot-1 and CAP may vary according to the signaling receptor and the respective cellular response. Impor- tantly, strong evidence connects all three proteins to MAPK activation, and FRS2 has been suggested to act as a scaffolder during MAPK signaling [1,14,34,38,47,48,49]. Our unpublished results imply that during EGF receptor signaling, flot-1 is found in complex with RAF, MEK and ERK kinases, suggesting a scaffolding function (Amaddii et al, under revision). Very interestingly, we identified another putative MAP kinase scaf- folder, mitogen activated protein kinase organizer-1, MORG1 [50,51] as a further interaction partner of flot-1 in our yeast two- hybrid screen (our unpublished results). Thus, the most likely mechanism how the interaction of FRS2 with flot-1/CAP modulates signal transduction is the regulation of MAP kinase function and the cellular activities resulting from MAP kinase activity, such as proliferation and differentiation. Materials and Methods Plasmid constructs FRS2 cDNA (NM_006654.3) was obtained by reverse tran- scription using the primers: 59-CTATAGAATTCATGGGTAGCTGTTGTAGCTG-39 59-CTATAAGATCTAGCATGGGCAGATCAGTACTATT- G-39 and cloned into vectors pECFP-N1 or pGADT7 (Clontech). A sequence encoding the PTB domain of FRS2 was generated by PCR using the primers 59-CTATAGAATTCATGGGTAGCTGTTGTAGCTG-39 59-CTATAAGATCTTCATATACTATTATTTTGCATAAT CTC-39 and the C-terminal region with the primers 59-CTATAGAATTCAATGTGGTGGAAGAGCCAGTTG-39 59-CTATAAGATCTAGCATGGGCAGATCAGTACTATT- G-39 GST-fusion constructs of FRS2 were constructed by subcloning into vector pGEX-4T-1 (GE Healthcare), and FRS2-His was PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e29739