FRS2 Interaction with Flotillin-1 and CAP Figure 6. FRS2 directly interacts with Cbl-associated protein. (A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 mg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2. doi:10.1371/journal.pone.0029739.g006 Flot-1 does not contain any typical PTB domain binding motifs (NPXY or NPXpY) although its interaction with FRS2 is to the most part mediated by the FRS2 PTB domain. Thus, other binding motifs must be responsible for the interaction. Consistently, the PTB domain of FRS2 can bind to both phosphorylated and nonpho- sphorylated sequences. It has been shown to interact with the Trk A receptor by means of a classical NPXpY motif, whereas its interaction with the juxtamembrane region of FGF receptor 1 involves an amino acid sequence lacking both Tyr and Asn residues [5,6,41]. However, Tyr phosphorylation was found to increase the binding of the full-length FRS2 but not of the PTB domain to FGFR1 [42]. We here observed an increased Tyr phosphorylation of FRS2 in the absence of flot-1. Previous studies have shown that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated [14,15,39]. Unfortunately, we were not able to measure FRS2 Thr phosphorylation, which is mediated by ERK, but it is likely to be reduced. This may even be a direct consequence of the reduced ERK activity in flot-1 knockdown cells, since inhibition of ERK activity has been shown to result in decreased Thr and concomitantly increased Tyr phosphorylation of FRS2 [15]. Another novel interaction partner of FRS2 discovered in the present study is CAP, an adaptor protein with three SH3 domains. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29739