RESUMO
Gene expression reprogramming governs cellular processes such as proliferation, differentiation and cell migration through the complex and tightly regulated control of transcriptional cofactors that exist in multiprotein complexes. Here we describe SCAI (suppressor of cancer cell invasion), a novel and highly conserved protein that regulates invasive cell migration through three-dimensional matrices. SCAI acts on the RhoA-Dia1 signal transduction pathway and localizes in the nucleus, where it binds and inhibits the myocardin-related transcription factor MAL by forming a ternary complex with serum response factor (SRF). Genome-wide expression analysis surprisingly reveals that one of the strongest upregulated genes after suppression of SCAI is beta1-integrin. Decreased levels of SCAI are tightly correlated with increased invasive cell migration, and SCAI is downregulated in several human tumours. Functional analysis of the beta1-integrin gene strongly argues that SCAI is a novel transcriptional cofactor that controls gene expression downstream of Dia1 to dictate changes in cell invasive behaviour.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Integrina beta1/genética , Invasividade Neoplásica , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Estruturas Animais/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Elementos Facilitadores Genéticos/genética , Expressão Gênica/genética , Humanos , Integrina beta1/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica/fisiologia , RNA Interferente Pequeno/genética , Homologia de Sequência de Aminoácidos , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismoRESUMO
Small GTPases of the Rho family are important modulators of the cytoskeleton and regulate morphogenetic cell movements during embryonic development. In the Xenopus embryo, Rho signaling contributes to the regulation of convergent extension (CE) movements in gastrula and neurula stages as well as to tissue separation (TS). Here we describe a method that allows the detection of activated (GTP-bound) Rho in fixed Xenopus tissue. The assay makes use of a fusion protein of Rhotekin and Green-Fluorescent-Protein (RBD-GFP), which is produced in bacteria and can be purified biochemically. This technique allows a temporal and spatial analysis of Rho signaling in the developing embryo. Developmental Dynamics 238:1407-1411, 2009. (c) 2009 Wiley-Liss, Inc.
Assuntos
Imuno-Histoquímica/métodos , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologia , Proteínas rho de Ligação ao GTP/genéticaRESUMO
SH4 domains provide bipartite membrane-targeting signals for oncogenic Src family kinases. Here we report the induction of non-apoptotic plasma membrane (PM) blebbing as a novel and conserved activity of SH4 domains derived from the prototypic Src kinases Src, Fyn, Yes and Lck as well as the HASPB protein of Leishmania parasites. SH4-domain-induced blebbing is highly dynamic, with bleb formation and collapse displaying distinct kinetics. These reorganizations of the PM are controlled by Rho but not Rac or Cdc42 GTPase signalling pathways. SH4-induced membrane blebbing requires the membrane association of the SH4 domain, is regulated by the activities of Rock kinase and myosin II ATPase, and depends on the integrity of F-actin as well as microtubules. Endogenous Src kinase activity is crucial for PM blebbing in SH4-domain-expressing cells, active Src and Rock kinases are enriched in SH4-domain-induced PM blebs, and PM blebbing correlates with enhanced cell invasion in 3D matrices. These results establish a novel link between SH4 domains, Src activity and Rho signalling, and implicate SH4-domain-mediated PM dynamization as a mechanism that influences invasiveness of cells transformed by SH4-domain-containing oncoproteins.
Assuntos
Motivos de Aminoácidos , Membrana Celular , Movimento Celular/fisiologia , Extensões da Superfície Celular , Quinases da Família src/metabolismo , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/ultraestrutura , Cricetinae , Cricetulus , Células HeLa , Humanos , Leishmania/metabolismo , Leishmania/patogenicidade , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genéticaRESUMO
The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Linhagem Celular Tumoral , Retroalimentação , Forminas , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Modelos Biológicos , Mutação , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Scratch-wound assays are frequently used to study directed cell migration, a process critical for embryogenesis, invasion, and tissue repair. The function and identity of trimeric G-proteins in cell behavior during wound healing is not known. Here we show that Galpha12/13, but not Galphaq/11 or Galphai, is indispensable for coordinated and directed cell migration. In mouse embryonic fibroblasts endogenous Rho activity is present at the rear of migrating cells but also at the leading edge, whereas it is undetectable at the cell front of Galpha12/13-deficient mouse embryonic fibroblasts. Spatial activation of Rho at the wound edge can be stimulated by lysophosphatidic acid. Active Rho colocalizes with the diaphanous-related formin Dia1 at the cell front. Galpha12/13-deficient cells lack Dia1 localization to the wound edge and are unable to form orientated, stable microtubules during wound healing. Knock down of Dia1 reveals its requirement for microtubule stabilization as well as polarized cell migration. Thus, we identified Galpha12/13-proteins as essential components linking extracellular signals to localized Rho-Dia1 function during directed cell movement.
