RESUMO
Cholesterol and sphingolipid-rich membrane microdomains or rafts have been shown to be involved in signaling through many growth factor receptors but the molecular details of these processes are not well understood. The reggie/flotillin proteins are ubiquitously expressed proteins with a poorly characterized function. They are constitutively associated with membrane rafts by means of acylation and oligomerization. Previous studies have implicated reggies in signaling, regulation of actin cytoskeleton and in membrane transport processes. In this study, we analyzed the putative role of reggie-1/flotillin-2 in signaling through the epidermal growth factor receptor. We show that reggie-1 becomes phosphorylated by Src kinase at several tyrosines upon stimulation of cells with epidermal growth factor. In addition, Src and reggie-1 are present as a molecular complex. Epidermal growth factor stimulation of cells results in a Tyr163-dependent translocation of reggie-1 from the plasma membrane into endosomes. We also show that reggie-1 is capable of enhancing the spreading of cells, again in a tyrosine-dependent manner, and knockdown of reggie-1 interferes with spreading. Thus, we reveal a new function for reggie-1 in the regulation of cell adhesion and actin dynamics and in growth factor signaling.
Assuntos
Adesão Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Membrana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Endossomos/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Células PC12 , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Tirosina/química , Quinases da Família src/metabolismoRESUMO
The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.
