RESUMO
The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G1 tetraploids in the population of dying E4orf4-expressing cells.
Assuntos
Mitose/fisiologia , Imagem Molecular/métodos , Proteínas Virais/fisiologia , Proteínas Virais/ultraestrutura , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imagem com Lapso de TempoRESUMO
The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.
Assuntos
Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/fisiopatologia , Adenovírus Humanos/metabolismo , Apoptose , Replicação do DNA , Pontos de Checagem da Fase G1 do Ciclo Celular , Proteína Supressora de Tumor p53/deficiência , Proteínas Virais/metabolismo , Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/genética , Animais , Morte Celular , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Diploide , Técnicas de Inativação de Genes , Humanos , Tetraploidia , Proteína Supressora de Tumor p53/genética , Proteínas Virais/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/fisiologia , Mitose , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Animais , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Ensaios de Triagem em Larga Escala , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.