Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling.
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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ABSTRACT: This review describes lipid raft ion channel complexes and EGFR in cancer cells•Complexes are composed of Ca2 + -activated K + (KCa), Cl- (ClCa) and Ca2 + channels•The modification of these lipid raft complexes by lipids is presented•This could lead to a novel therapeutic approach in tumor developmentBiochimica et Biophysica Acta (BBA) - Biomembranes. 11/2014;
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ABSTRACT: The RAF family of kinases mediates RAS signaling, and RAF inhibitors can be effective for treating tumors with BRAF(V600E) mutant protein. However, RAF inhibitors paradoxically accelerate metastasis in RAS-mutant tumors and become ineffective in BRAF(V600E) tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling. We found that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and cell migration in a cell type-dependent manner. Knocking down ARAF prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and decreased the number of protrusions from tumor cell spheroids in three-dimensional culture that were induced by BRAF(V600E)-specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors induced the homodimerization of ARAF and the heterodimerization of BRAF with CRAF and the scaffolding protein KSR1. In a purified protein solution, recombinant proteins of the three RAF isoforms competed for binding to MEK1. In cells in culture, overexpressing mutants of ARAF that could not homodimerize impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent activation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade and promoting tumor cell invasion and suggest a new therapeutic target for RAS- and RAF-mediated cancers.Science Signaling 01/2014; 7(337):ra73. · 7.65 Impact Factor
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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 01/2014; 9(9):e106330. · 3.53 Impact Factor