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. 2012;7(1):e29739.
doi: 10.1371/journal.pone.0029739. Epub 2012 Jan 3.

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Ana Tomasovic  1 , Stephanie Traub, Ritva Tikkanen
Affiliations

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Ana Tomasovic et al. PLoS One. 2012.

Abstract

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification of interaction domains in FRS2 and flot-1.
(A) Yeast two-hybrid analysis of the interaction between FRS2 and flot-1 domains. Interaction is indicated as growth of blue colonies on nutrient deficient plates containing α-X-galactoside. (B) FRS2 domains were produced as GST fusion proteins, immobilized on glutathione beads and tested for the interaction with endogenous flot-1 from HeLa cell lysates. Upper blot: detection of bound flot-1, lower blot: ponceau staining of the respective GST proteins. (C) FL flot-1 or its C-terminal half were produced as GST fusion proteins, immobilized on beads and incubated with purified FRS2-His. Upper blot: detection of bound FRS2-His, lower blot: ponceau staining of the purified GST proteins. Specific bands for the GST fusions are marked with *. (D) FRS2 was immunoprecipitated from lysates of 25 mg of mouse tissue (brain, liver and kidney), and the coprecipitation of flot-1 was studied. Antibody against flag tag (IgG control) was used as a control for the immunoprecipitation. Right part shows a blot for FRS2 with total tissue lysates (equal total protein amount).
Figure 2
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 µm.
Figure 3
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 µg). 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.
Figure 4
Figure 4. Increased Tyr phosphorylation and solubility of FRS2 in flotillin knockdown cells.
(A) FRS2 was immunoprecipitated from serum grown Hep3B cells. The Tyr phosphorylation of FRS2 was measured by means of phospho-Tyr antibodies and found to be increased both in flot-1 and flot-2 knockdown cells. (B) Densitometric quantification of FRS2 phosphorylation with SD (5 independent experiments). F1-KD cells display a significantly increased P-Tyr of FRS2. (C) Hep3B cells were grown under serum, fixed and stained with antibodies against FRS2 (left column) and flotillins (middle). In control cells, FRS2 was localized at the plasma membrane and within the cytosol, whereas in flot-1 or flot-2 knockdown cells, a cytosolic staining was evident. In addition, especially in flot-2 knockdown, some nuclear staining was observed. Right column: overlay with DAPI staining. Scale bars 10 µm.
Figure 5
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.
Figure 6
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 µg 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.
Figure 7
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 µg) 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.
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