RESUMEN
α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.
Asunto(s)
Quimerina 1/química , Quimerina 1/metabolismo , Diglicéridos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Neuronas/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Ubiquitina/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.
Asunto(s)
Quimerina 1/química , Proteínas Activadoras de GTPasa/química , Proteínas de Unión al GTP rac/metabolismo , Animales , Células COS , Chlorocebus aethiops , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Humanos , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Fosfolipasas de Tipo C/químicaRESUMEN
Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes alpha2-chimaerin, a Rac guanosine triphosphatase-activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase alpha2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance alpha2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant alpha2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that alpha2-chimaerin has a critical developmental function in ocular motor axon pathfinding.
Asunto(s)
Quimerina 1/genética , Quimerina 1/metabolismo , Síndrome de Retracción de Duane/genética , Mutación Missense , Nervio Abducens/anomalías , Secuencia de Aminoácidos , Animales , Axones/fisiología , Línea Celular , Membrana Celular/metabolismo , Embrión de Pollo , Quimerina 1/química , Femenino , Perfilación de la Expresión Génica , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Músculos Oculomotores/embriología , Músculos Oculomotores/inervación , Músculos Oculomotores/metabolismo , Nervio Oculomotor/anomalías , Nervio Oculomotor/embriología , LinajeRESUMEN
EphA4-dependent growth cone collapse requires reorganization of actin cytoskeleton through coordinated activation of Rho family GTPases. Whereas various guanine exchange factors have recently been identified to be involved in EphA4-mediated regulation of Rho GTPases and growth cone collapse, the functional roles of GTPase-activating proteins in the process are largely unknown. Here we report that EphA4 interacts with alpha2-chimaerin through its Src homology 2 domain. Activated EphA4 induces a rapid increase of tyrosine phosphorylation of alpha2-chimaerin and enhances its GTPase-activating protein activity toward Rac1. More importantly, alpha2-chimaerin regulates the action of EphA4 in growth cone collapse through modulation of Rac1 activity. Our findings have therefore identified a new alpha2-chimaerin-dependent signaling mechanism through which EphA4 transduces its signals to the actin cytoskeleton and modulates growth cone morphology.