RESUMEN
Despite the fact that inositol hexakisphosphate (InsP(6)) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP(6). The substrate for the InsP(6)-regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP(6)-dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP(6)-dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/metabolismo , GTP Fosfohidrolasas/metabolismo , Hipocampo/metabolismo , Proteínas Quinasas/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas del Citoesqueleto , Dinamina I , Dinaminas , Endocitosis , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Ácido Fítico , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The PACSINs are a family of cytoplasmic phosphoproteins that play a role in vesicle formation and transport. We report the cloning and cDNA sequencing of PACSIN 3 and the analysis of all three PACSIN isoforms with regard to tissue distribution, ligand binding properties and influence on endocytosis. PACSIN 3 differs from the other family members in having a short proline-rich region and lacking asparagine-proline-phenylalanine motifs. In contrast to the neurospecific PACSIN 1 and the ubiquitously expressed PACSIN 2, PACSIN 3 is mainly detected in lung and muscle tissues. All isoforms potentially oligomerize and bind to dynamin, synaptojanin 1 and N-WASP via their Src homology 3 domains. The PACSIN proteins colocalize with dynamin, but not with clathrin, implying a specific role with a distinct subpopulation of dynamin at defined cellular sites. Transferrin endocytosis is blocked in a dose-dependent manner in cells overexpressing the PACSIN variants, but the inhibitory effect can be abolished by mutating specific amino acid residues in the Src homology 3 domains. These characteristics of the PACSIN protein family suggest a general function in recruitment of the interacting proteins to sites of endocytosis.
Asunto(s)
Endocitosis/fisiología , GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas del Citoesqueleto , ADN Complementario , Dinaminas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Fosfoproteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Distribución Tisular , Transferrina/metabolismo , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
The PACSIN-related proteins are cytoplasmic adapter proteins with a common arrangement of domains and conserved regions. Here we report the cloning, sequencing, and expression of PACSIN 2, a novel member of the PACSIN protein family and accordingly rename the original PACSIN to PACSIN 1. The sequences of the murine and human cDNAs reveal an open reading frame encoding a putative protein of 486 residues. Despite its high sequence similarity to PACSIN 1, PACSIN 2 is encoded by distinct transcripts in human and mouse, in particular displaying a ubiquitous expression pattern. Immunofluorescence microscopy of PACSIN 2-transfected NIH3T3 fibroblasts reveal a broad, vesicle-like cytoplasmic staining. In contrast to FAP52, another PACSIN-related protein derived from chicken brain, PACSIN 2 could not be detected at focal contacts. Taken together, these findings suggest that PACSIN 2 is a novel PACSIN isoform with similar domain and motif arrangement, but an unrestricted expression pattern, which may participate in the organization of the actin cytoskeleton and the regulation of vesicular traffic.