RESUMO
The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Fibroblastos/fisiologia , Proteínas Motores Moleculares/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Carbazóis/farmacologia , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Camundongos , Proteínas Motores Moleculares/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Propanolaminas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an integral membrane protein that functions as a constitutively activated member of the tumor necrosis factor receptor family. Whereas LMP1 has been shown to activate the NF-kappaB and mitogen-activated protein kinase pathways, these effects alone are unable to account for the profound oncogenic properties of LMP1. Here we show that LMP1 can activate phosphatidylinositol 3-kinase (PI3K), a lipid kinase responsible for activating a diverse range of cellular processes in response to extracellular stimuli. LMP1 was found to stimulate PI3K activity inducing phosphorylation and subsequent activation of Akt, a downstream target of PI3K responsible for promoting cell survival. Treatment of LMP1-expressing cells with the PI3K inhibitor LY294002 resulted in decreased cell survival. The tumor necrosis factor receptor-associated factor-binding domain of LMP1 was found to be responsible for PI3K activation. The ability of LMP1 to induce actin stress-fiber formation, a Rho GTPase-mediated phenomenon, was also dependent on PI3K activation. These data implicate PI3K activation in many of the LMP1-induced phenotypic effects associated with transformation and suggests that this pathway contributes both to the oncogenicity of this molecule and its role in the establishment of persistent EBV infection.
