RESUMEN
We have reported previously that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MyoGEF) plays an important role in the regulation of cell migration and cytokinesis. Like many other guanine nucleotide exchange factors (GEFs), MyoGEF contains a Dbl homology (DH) domain and a pleckstrin homology domain. In this study, we provide evidence demonstrating that intramolecular interactions between the DH domain (residues 162-351) and the carboxyl-terminal region (501-790) of MyoGEF can inhibit MyoGEF functions. In vitro and in vivo pulldown assays showed that the carboxyl-terminal region (residues 501-790) of MyoGEF could interact with the DH domain but not with the pleckstrin homology domain. Expression of a MyoGEF carboxyl-terminal fragment (residues 501-790) decreased RhoA activation and suppressed actin filament formation in MDA-MB-231 breast cancer cells. Additionally, Matrigel invasion assays showed that exogenous expression of the MyoGEF carboxyl-terminal region decreased the invasion activity of MDA-MB-231 cells. Moreover, coimmunoprecipitation assays showed that phosphorylation of the MyoGEF carboxyl-terminal region by aurora B kinase interfered with the intramolecular interactions of MyoGEF. Furthermore, expression of the MyoGEF carboxyl-terminal region interfered with RhoA localization during cytokinesis and led to an increase in multinucleation. Together, our findings suggest that binding of the carboxyl-terminal region of MyoGEF to its DH domain acts as an autoinhibitory mechanism for the regulation of MyoGEF activation.
Asunto(s)
Citocinesis/genética , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Línea Celular Tumoral , Movimiento Celular , Colágeno/química , Combinación de Medicamentos , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Laminina/química , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteoglicanos/química , Transducción de Señal , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.
Asunto(s)
Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocinesis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Treonina/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Inmunoprecipitación , Mitosis , Fosforilación , Unión Proteica , Huso Acromático/metabolismo , Treonina/genética , Quinasa Tipo Polo 1RESUMEN
The mammalian neocortex has undergone remarkable changes through evolution. A consequence of such rapid evolutionary events could be a trade-off that has rendered the brain susceptible to certain neurodevelopmental and neuropsychiatric conditions. We analyzed the exomes of 65 patients with the structural brain malformation periventricular nodular heterotopia (PH). De novo coding variants were observed in excess in genes defining a transcriptomic signature of basal radial glia, a cell type linked to brain evolution. In addition, we located two variants in human isoforms of two genes that have no ortholog in mice. Modulating the levels of one of these isoforms for the gene PLEKHG6 demonstrated its role in regulating neuroprogenitor differentiation and neuronal migration via RhoA, with phenotypic recapitulation of PH in human cerebral organoids. This suggests that this PLEKHG6 isoform is an example of a primate-specific genomic element supporting brain development.