RESUMO
Invasive cell migration is a key step for cancer metastasis and involves Rho GTPase-controlled reorganization of the actin cytoskeleton. Altered Rho GTPase expression is found in various malignancies. Particularly, the closely related GTPases RhoA and RhoC are upregulated in many aggressive tumours, but specific effectors that distinguish between these two GTPases to explain mechanistic differences have not been identified. The formins are by far the largest family of Rho GTPase effectors and are characterized by the actin-nucleating formin homology 2 domain. Using siRNA-based screening against all 15 human formins, we systematically analysed their functions in 3D cell motility using three different cancer cell lines. These results reveal distinct requirements for specific formins in amoeboid versus mesenchymal invasive cell migration. Importantly, by knocking down all Rho proteins, we identified formin-like 2 (FMNL2) as a specific RhoC effector, showing selective interaction of FMNL2 with active RhoC, but not RhoA or RhoB. Functional analysis shows that RhoC regulates autoinhibition of FMNL2, whereas suppression of FMNL2 inhibits RhoC-, but not RhoA-dependent, rounded invasive cell migration. Thus, our data uncover a novel regulatory and functional interaction between RhoC and FMNL2 for modulating cell shape and invasiveness and provide mechanistic insight into RhoC-specific signalling events.
Assuntos
Invasividade Neoplásica , Proteínas/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Forminas , Humanos , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteína de Ligação a GTP rhoCRESUMO
To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Sequência Conservada , DNA/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila , Evolução Molecular , Fatores de Transcrição Fushi Tarazu , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Proteínas de Insetos/genética , Ligantes , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/genética , Homologia de Sequência de Aminoácidos , Fator Esteroidogênico 1 , Fatores de Transcrição/genéticaRESUMO
Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Feminino , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hormônios de Inseto/genética , Proteínas de Insetos , Masculino , Mutagênese , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Receptores Citoplasmáticos e Nucleares/genética , Fator Esteroidogênico 1 , Fatores de Transcrição/genética , Proteína Wnt1RESUMO
Homeodomain proteins regulate diverse developmental processes in a wide range of organisms, yet bind in vitro to DNA sequences that are remarkably similar. This has raised the fundamental question of how target gene specificity is achieved in vivo. The Drosophila fushi tarazu protein (Ftz) contains a homeodomain and is required for the formation of alternate segments. We have shown previously that a homeodomain-deleted Ftz polypeptide (Ftz delta HD), incapable of binding DNA in vitro, could regulate endogenous ftz gene expression. Here we test Ftz delta HD activities in a ftz mutant background and find that, surprisingly, Ftz delta HD can directly regulate ftz-dependent segmentation, suggesting that it can control target gene expression through interactions with other proteins. A likely candidate is the pair-rule protein Paired (Prd). Ftz delta HD bound directly to Prd in vitro and required Prd to repress wingless in vivo. These results emphasize the pivotal importance of protein-protein interactions in homeodomain protein function.
