Open Access

Ana Tomasovic

• The long sought molecular function of membrane raft-associated flotillin proteins is slowly becoming resolved, partially owing to the increasing knowledge about their interaction partners. Being ubiquitously expressed and evolutionarily highly conserved, flotillins carry out important cellular functions, one of which is the regulation of signal transduction pathways. This study shows that the signaling adaptor protein fibroblast growth factor receptor substrate 2 (FRS2) directly interacts both in vivo and in vitro with flotillin-1 (flot-1). FRS2 is an important docking protein of many receptor tyrosine kinases. It regulates downstream signaling by forming molecular complexes with other adaptor proteins and tyrosine phosphatases, and seems to be a critical mediator of sustained extracellular signal regulated kinase (ERK) activity. Flot-1 has also been implicated in the regulation of ERK activity upon EGF and FGF stimuli. Furthermore, flot-1 forms signalosomes with EGFR and the downstream components of the MAP kinase pathway. The newly discovered interaction between FRS2 and flot-1 was shown to be mediated by the phosphotyrosine binding (PTB) domain and, to a lesser extent, the C-terminus (CT) of FRS2 and by the C-terminus of flot-1. Flot-1 coprecipitated together with FRS2 from murine tissues and cell lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it, does not appear to directly interact with FRS2. Novel insights into the functional role of the interaction between flot-1 and FRS2 were provided by the results showing that depletion of flot-1 affects the cellular localization of FRS2. In hepatocytes stably depleted of flot-1, FRS2 appeared to be more soluble. Furthermore, upon pervanadate stimulation of the cells, a small fraction of FRS2 was recruited into detergent resistant membranes, but the recruitment did not take place in the absence of flot-1. Triggered by the same stimulus, a fraction of FRS2 was translocated to the nucleus independently of flot-1. Overexpression of FRS2 has previously been shown to result in increased ERK activation. However, in cells depleted of flot-1, FRS2 was not able to compensate for the compromised ERK activation after EGF or FGF stimulation. This might imply that FRS2 and flot-1 are functionally interconnected and that FRS2 resides upstream of flot-1. Taken together, the results presented here indicate that this complex may be involved in the control of signaling downstream of receptor tyrosine kinases and is important for ensuring a proper signaling response. In the absence of flot-1, increased Tyr phosphorylation of FRS2 was observed. It is known that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated. Since ERK is a known executor of the FRS2 Thr phosphorylation, and ERK activity was shown to be severely diminished upon flot-1 depletion, the increased Tyr phosphorylation of FRS2 was in agreement with this and might be a direct consequence of a decreased ERK activity upon flot-1 depletion. FRS2 owes its name to the major and the first described function of this protein as a substrate for FGFR. PTB domain of FRS2 was published to constitutively bind the juxtamembrane domain of FGFR. In this study, the PTB domain was mapped to be involved in the constitutive interaction with flot-1 and the competition was shown to exist between flot-1 and FGFR1 for binding to FRS2. Another novel interaction partner of FRS2 was discovered in the present study. Cbl-associated protein (CAP) is an adaptor protein with three SH3 domains and it plays a role during insulin signaling by recruiting the signaling complex to lipid rafts. CAP was previously shown to interact with flot-1 via the SoHo domain, and this interaction was found to be crucial for the lipid raft recruitment of other signaling components. Both the PTB domain and CT of FRS2 were found to mediate the interaction with CAP, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. SH3 domains mediate the assembly of specific protein complexes by binding to proline rich sequences, several of which are present in FRS2. Due to overlapping interaction domains, FRS2 and flot-1 competed for the binding to CAP. However, the interaction with neither CAP nor flot-1 was necessary for the observed nuclear translocation of FRS2. Since CAP is expressed as several tissue- and developmental stage-specific isoforms, a further aim of this study was to analyze the expression of its isoforms in mouse embryonic fibroblasts (MEFs). Many new isoforms were discovered here which have not been described in the literature so far. They all contain the SoHo domain and three SH3 domains, but differ among themselves by the presence and length of a proline-rich region that preceeds the SoHo domain and by a novel 20-amino acid (AA) stretch between the second and the third SH3 domain. The length of the proline-rich region turned out to be an important factor determining the strength of the interaction with FRS2. The interaction was found to be weakened by the increasing length of this region. The new isoforms possessing the 20-AA stretch are specifically expressed in murine muscular tissues, with the highest level in the heart. During adipogenesis, we observed a shift in the abundance of the isoforms, in that only the isoforms without the insertion were shown to be upregulated on mRNA level. However, during myogenesis, preferentially expressed isoforms were those with the insertion. The collected data implicate that isoforms with the 20-AA insertion might be more ubiquitous in nondifferentiated/embryonic cells and that the observed "isoform-switch" might be dependent on the cell fate and differentiation state.
• FRS2 (engl. fibroblast growth factor receptor substrate 2) ist ein Membran-gebundenes Adapterprotein, welches Signalkaskaden ausgehend von verschiedenen Rezeptor-Tyrosinkinasen reguliert. Die molekulare Struktur von FRS2 ist identisch mit dem homologen Protein FRS3. FRS2 beinhaltet eine Myristoylierungssequenz in der Nähe des N-Terminus, eine Phosphotyrosin-Bindedomäne (PTB, engl. phosphotyrosine binding domain) und multiple Tyrosin-Phosphorylierungsstellen im Bereich des CTerminus. Namensgebend für die Proteine der FRS-Familie ist ihre essentielle Funktion bei der Signaltransduktion ausgehend vom FGF-Rezeptor, da dieser selbst keine Adapterproteine rekrutieren kann, die das Signal an MAP-Kinasen weiterleiten. Die Phosphorylierung von FRS2, welche nach Stimulation mit NGF, BDNF, FGF und GDNF erfolgt, ermöglicht die Bildung von Signalkomplexen mit weiteren Adapterproteinen (z.B. Grb2 und SOS) und der Tyrosinphosphatase Shp2. Die Rekrutierung dieser Komponenten resultiert in der Aktivierung der MAP-Kinase- und der PI3K/Akt-Signalkaskade. Auf Grund seiner regulatorischen Funktion nach Stimulierung mit FGF oder NGF, spielt FRS2 eine wichtige Rolle bei der Differenzierung von PC12 Zellen. ... In dieser Studie wurden Flotillin-1 und CAP als neue Interaktionspartner von FRS2 identifiziert. Flotillin-1 bindet an die PTB Domäne von FRS2, wodurch es mit dem FGFR um dessen Bindung konkurriert. Darüber hinaus führt die Deletion von Flotillin-1 zu einer verstärkten Tyrosinphosphorylierung von FRS2. Dieses Ergebnis stimmt mit der beobachteten Inhibierung der ERK Aktivität in Abwesenheit von Flotillin- 1 überein. Das Protein CAP bindet ebenfalls direkt an FRS2, wobei diese Interaktion von der dritten SH3- Domäne und der SoHo-Domäne von CAP, sowie der PTB-Domäne und dem C-Terminus von FRS2 vermittelt wird. Sie SoHo-Domäne ist zusätzlich verantwortlich für die Interaktion von CAP mit Flotillin-1, was impliziert das auch CAP mit Flotillin-1 um die Bindung von FRS2 konkurriert. Dieses neu entdeckte Proteinnetzwerk könnte eine wichtige Rolle bei der Regulation der Signaltransduktion ausgehend von Rezeptor-Tyrosinkinasen spielen. Desweiteren wurde in dieser Studie nachgewiesen, dass verschiedene CAP Isoformen in MEFs exprimiert werden. All diese Isoformen beinhalten eine SoHo-Domäne und SH3-Domänen, unterscheiden sich jedoch im Vorkommen und der Länge einer Prolin-reichen Sequenz und einer neu entdeckten 20 Aminosäure-langen Insertion. Isoformen, welche diese Insertion besitzen, wurden hauptsächlich in murinen Muskelgewebe nachgewiesen - mit höchster Expression im Herzen. Die Isoformen ohne Insertion werden auf mRNA-Level während des Vorgangs der Adipogenese hochreguliert. Für Isoformen ohne Insertion konnte auf mRNA-Level keine Veränderung detektiert werden. Diese Ergebnisse lassen vermuten, dass die neuen murinen CAP Isoformen mit Insertion eine Rolle während der Entwicklung des Herzens spielen.