RESUMEN
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
Asunto(s)
Citoesqueleto/genética , Proteínas Activadoras de GTPasa/genética , Integrinas/genética , Mecanotransducción Celular/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína de Unión al GTP rac1/genética , Animales , Células COS , Adhesión Celular , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Biología Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Perros , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Pan troglodytes , Dominios Proteicos , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/clasificación , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/fisiología , Proteínas de Fusión Oncogénica/metabolismo , Dominios Proteicos/fisiología , Proteína que Contiene Valosina/metabolismo , Encéfalo/patología , Proliferación Celular , Cristalografía por Rayos X , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/aislamiento & purificación , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Ingeniería de Proteínas , Mapas de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína que Contiene Valosina/química , Proteína que Contiene Valosina/aislamiento & purificaciónRESUMEN
Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.