Molecular Networks in FGF Signaling: Flotillin-1 and Cbl- Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2 Ana Tomasovic1,2, Stephanie Traub2¤, Ritva Tikkanen1,2* 1 Institute of Biochemistry, University of Giessen, Giessen, Germany, 2 Institute of Biochemistry II, University Clinic of Frankfurt, Frankfurt am Main, Germany Abstract Fibroblast growth factor receptor substrate 2 (FRS2a) 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. Citation: Tomasovic A, Traub S, Tikkanen R (2012) Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2. PLoS ONE 7(1): e29739. doi:10.1371/journal.pone.0029739 Editor: Henning Ulrich, University of Sa˜o Paulo, Brazil Received July 12, 2011; Accepted December 4, 2011; Published January 3, 2012 Copyright: 2012 Tomasovic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funds were received from Deutsche Forschungsgemeinschaft DFG (www.dfg.de), personal grant to RT (Ti 291/6-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Ritva.Tikkanen@biochemie.med.uni-giessen.de ¤ Current address: Dermatology, University Clinic of Cologne, Cologne, Germany Introduction Fibroblast growth factor receptor substrate 2 (FRS2/FRS2a/ SNT1) is a membrane linked docking protein originally identified as a protein that becomes tyrosine phosphorylated upon nerve growth factor (NGF) or fibroblast growth factor (FGF) stimulation in PC12 cells [1,2,3]. Together with FGF receptor substrate 3 (FRS3/FRS2b/SNT2), it belongs to the FRS adaptor protein family [4]. In this paper, we will use the name FRS2 for FRS2a/ SNT1, and FRS3 for FRS2b/SNT2 for the sake of clarity. FRS2 and FRS3 share a similar structure and 48% of amino acid sequence identity. In the N-terminus, they contain a consensus myristoylation sequence which is important for the membrane localization [1]. This sequence is followed by a phosphotyrosine binding (PTB) domain that is highly similar between the two proteins. The PTB domain binds specific peptides of certain receptor tyrosine kinases (RTKs) with or without tyrosine phosphorylated residues [5,6]. FRS2 is ubiquitously expressed with the highest expression in brain, kidney, lung, ovary and testis and can be detected at all developmental stages of a mouse [7]. FRS2 knockout mice show embryonic lethality due to severe problems in gastrulation [8,9], demonstrating how crucial FGF signaling is in animal development. In contrast to FRS2, the expression of FRS3 protein begins around day 9 and is restricted to tissues of neuronal origin [7,10]. When exogenously expressed in FRS2-null mouse embryonic fibroblasts, FRS3 compensates for the loss of FRS2 by stimulating FGF induced activation of extracellularly regulated kinase (ERK), a member of the mitogen activated protein (MAP) kinase family [10]. Both FRS2 and FRS3 are tyrosine phosphorylated in response to NGF, FGF [9,11] and glial derived neurotrophic factor (GDNF) [12,13], but only FRS2 is threonine phosphorylated. Phosphor- ylation of 8 threonine residues in FRS2 occurs as a response to stimulation with FGF, epidermal growth factor (EGF), insulin and platelet derived growth factor (PDGF). This represents a negative feedback mechanism in which activated ERK inhibits further tyrosine phosphorylation of FRS2 by phosphorylating its threo- nine residues [14,15]. FRS2 plays an important role in FGF dependent proliferation and migration of the cells [1] and in differentiation of PC12 cells by regulating sustained ERK activity upon FGF or NGF stimuli [1,16,17]. It is tyrosine phosphorylated upon insulin treatment, but its precise role in insulin signaling pathway remains largely unknown [18]. The C-terminus of FRS proteins bears multiple tyrosine phosphorylation sites (6 Tyr in FRS2), which, when phosphory- lated by specific RTKs, recruit SH2-domain containing proteins such as adaptor protein Grb2 (4 Tyr in FRS2) and protein tyrosine phosphatase Shp2 (2 Tyr in FRS2) [1,19]. The recruitment of Grb2 eventually results in a strong activation of PI3-kinase signaling and moderate activation of ERK pathway [9,20], while phosphorylation and subsequent activation of Shp2 will result in a strong activation of ERK signaling [9]. PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29739