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Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling

From the Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2012; 287(10):7265-78. DOI: 10.1074/jbc.M111.287599
Source: PubMed

ABSTRACT Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.

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    • "The flotillins form homo-and hetero-oligomers (Neumann-Giesen et al., 2004; Solis et al., 2007), which assemble into clusters defining specialized flat membrane microdomains at the plasma membrane independently of caveolins and caveolae (Fernow et al., 2007; Frick et al., 2007; Lang et al., 1998; Stuermer et al., 2001). Flotillins have been shown to be involved in various cellular processes including cell proliferation, T-cell activation and phagocytosis (Dermine et al., 2001; Gómez et al., 2010; Santamaría et al., 2005; Stuermer et al., 2004), and have been implicated in signaling activation of IgE and TrkA, and epidermal growth factor, insulin and G-protein-coupled receptors (Amaddii et al., 2012; Baumann et al., 2000; Kato et al., 2006; Limpert et al., 2007; Wehmeyer et al., 2014). Insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase activation leads to downstream signaling activation of the mitogenactivated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)–Akt pathways, which promote cell proliferation and differentiation and prevents apoptosis (Adams et al., 2000). "
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    ABSTRACT: Flotillin-1 regulation of insulin-like growth factor-1 (IGF-1) signaling was explored. Flotillin-1-deficient cells exhibited retardation in the activation of IGF-1 receptor (IGF-1R), ERK, Akt and Elk-1 transcription, and their proliferation in response to IGF-1. IGF-1-independent flotillin-1 palmitoylation at Cys-34 in endoplasmic reticulum (ER) was required for ER exit and plasma membrane (PM) targeting of flotillin-1 and IGF-1R. IGF-1-dependent depalmitoylation and repalmitoylation of the flotillin-1 sustained tyrosine kinase activation of the PM-targeted IGF-1R. Dysfunction and blocking the turnover of flotillin-1 palmitoylation abrogated cancer cell proliferation via IGF-1R signaling activation. Our data show that flotillin-1 palmitoylation presents novel mechanisms by which intracellular localization and activation of IGF-1R are controlled. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 04/2015; 128(11). DOI:10.1242/jcs.169409 · 5.33 Impact Factor
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    • "Flotillins play a major role in the fidelity of cell signaling and EGFR function [46]–[48]. Knockdown of flotillin-2 disrupts localization and phosphorylation of EGFR and activation of downstream MAPK signaling components [22]. However, in breast cancer cells with mutant PIK3CA, a well-known downstream target of EGFR signaling, knockdown of flotillin-1 causes an upregulation of EGFR and hyperactivation of MAPK signaling. "
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    ABSTRACT: Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations.
    PLoS ONE 09/2014; 9(9):e106330. DOI:10.1371/journal.pone.0106330 · 3.23 Impact Factor
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    • "Flotillins have been suggested to be involved in a plethora of cellular processes such as membrane receptor signaling, phagocytosis and endocytosis, cell-matrix adhesion and regulation of actin cytoskeleton [32]–[36]. Our recent data have revealed an important role for flotillin-1 as a regulator of epidermal growth factor receptor (EGFR) activation and as a scaffold protein for mitogen activated protein (MAP) kinase signaling [32]. Knockout mouse models for both flotillins have recently been generated, but they do not show any major developmental defects [37]–[39]. "
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    ABSTRACT: Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6-8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.
    PLoS ONE 12/2013; 8(12):e84393. DOI:10.1371/journal.pone.0084393 · 3.23 Impact Factor
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Melanie Meister