RESUMEN
Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular , Adhesiones Focales/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Proteínas Portadoras/genética , Adhesión Celular , Polaridad Celular , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Peróxido de Hidrógeno/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The actin cytoskeleton is critical for form and function of vascular cells, serving mechanical, organizational and signaling roles. Because many cytoskeletal proteins are sensitive to reactive oxygen species, redox regulation has emerged as a pivotal modulator of the actin cytoskeleton and its associated proteins. Here, we summarize work implicating oxidants in altering actin cytoskeletal proteins and focus on how these alterations affect cell migration, proliferation and contraction of vascular cells. Finally, we discuss the role of oxidative modification of the actin cytoskeleton in vivo and highlight its importance for vascular diseases.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/genética , Vasos Sanguíneos/metabolismo , Células Endoteliales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Vasos Sanguíneos/citología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , División Celular , Movimiento Celular , Células Endoteliales/ultraestructura , Regulación de la Expresión Génica , Humanos , Miosinas/genética , Miosinas/metabolismo , Oxidación-Reducción , Transducción de Señal , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Ras and Rho family GTPases control a wide variety of cellular processes, and the signaling downstream of these GTPases is influenced by their subcellular localization when activated. Since only a minority of total cellular GTPases is active, observation of the total subcellular distribution of GTPases does not reveal where active GTPases are localized. In this chapter, we describe the use of effector recruitment assays to monitor the subcellular localization of active Ras and Rho family GTPases. The recruitment assay relies on preferential binding of downstream effectors to active GTPases versus inactive GTPases. Tagging the GTPase-binding-domain (GBD) of a downstream effector with a fluorescent protein produces a probe that is recruited to compartments where GTPases are active. We describe an example of a recruitment assay using the GBD of PAK1 to monitor Rac1 activity and explain how the assay can be expanded to determine the subcellular localization of activation of other GTPases.
Asunto(s)
Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Línea Celular , Activación Enzimática , Espacio Intracelular/metabolismo , Imagen Molecular , Transporte de Proteínas , Coloración y Etiquetado , Transfección , Proteínas ras/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation.