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


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 · 5.43 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 seem to have a specific and regulatory role on ErbB receptors in different breast cancer cell lines. It has been shown by others that flotillin-depletion interferes with EGFR clustering [2] and EGFR phosphorylation [43] and affects downstream signaling cascades. Fig. 3. ErbB2 and ErbB3 receptor downregulation upon flotillin depletion. "
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    ABSTRACT: The ErbB3 receptor is an important regulator of cell growth and carcinogenesis. Among breast cancer patients, up to 50-70% have ErbB3 overexpression and 20-30% show overexpressed or amplified ErbB2. ErbB3 has also been implicated in the development of resistance to several drugs used against cancers driven by ErbB1 or ErbB2. One of the main challenges in ErbB-targeting therapy is to inactivate signaling mediated by ErbB2-ErbB3 oncogenic receptor complexes. We analyzed the regulatory role of flotillins on ErbB3 levels and ErbB2-ErbB3 complexes in SKBR3, MCF7 and MDA-MB-134-VI human breast cancer cells. Recently, we described a mechanism for interfering with ErbB2 signaling in breast cancer and demonstrated a molecular complex of flotillin scaffolding proteins with ErbB2 and Hsp90. In the present study, flotillins were found to be in a molecular complex with ErbB3, even in cells without the presence of ErbB2 or other ErbB receptors. Depletion of either flotillin-1 or flotillin-2 resulted in downregulation of ErbB3 and a selective reduction of ErbB2-ErbB3 receptor complexes. Moreover, flotillin-2 depletion resulted in reduced activation of Akt and MAPK signaling cascades, and as a functional consequence of flotillin depletion, breast cancer cells showed an impaired cell migration. Altogether, we provide data demonstrating a novel and functional role of flotillins in the regulation of ErbB protein levels and stabilization of ErbB2-ErbB3 receptor complexes. Thus, flotillins are crucial regulators for oncogenic ErbB function and potential targets for cancer treatment.
    Biochimica et Biophysica Acta 04/2014; 1843(9). DOI:10.1016/j.bbamcr.2014.04.013 · 4.66 Impact Factor
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Melanie Meister