Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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Molecular networks in signaling pathways: Flotillin-1, Cbl-associated protein and Fibroblast Growth Factor Receptor Substrate 2
(2012)
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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.
