RESUMO
Kalirin-7 (Kal7), a multifunctional Rho GDP/GTP exchange factor (GEF) for Rac1 and RhoG, is embedded in the postsynaptic density at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. Kal7 has been implicated in long-term potentiation, fear memories, and addiction-like behaviors. Using liquid chromatography and tandem mass spectroscopy, we identified sites phosphorylated by six PSD-localized kinases implicated in synaptic plasticity and behavior, sites phosphorylated when myc-Kal7 was expressed in non-neuronal cells and sites phosphorylated in mouse brain Kal7. A site in the Sec14p domain phosphorylated by calcium/calmodulin dependent protein kinase II, protein kinase A and protein kinase C, was phosphorylated in mouse brain but not in non-neuronal cells. Phosphorylation in the spectrin-like repeat region was more extensive in mouse brain than in non-neuronal cells, with a total of 20 sites identified. Sites in the pleckstrin homology domain and in the linker region connecting the GEF domain to the PDZ binding motif were heavily phosphorylated in both non-neuronal cells and in mouse brain and affected GEF activity. We postulate that the kinase convergence and divergence observed in Kal7 identify it as a key player in integration of the multiple inputs that regulate synaptic structure and function.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Densidade Pós-Sináptica/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transmissão Sináptica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Domínio Catalítico , Linhagem Celular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas em Tandem , Treonina/química , Treonina/genética , Treonina/metabolismoRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and ß2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4ß2-containing (α4ß2(∗)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4ß2(∗) nAChRs in a genedose dependent pattern by immunopurifying ß2(∗) nAChRs from mice that differ in α4 and ß2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the ß2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4ß2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a ß2(∗) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4ß2 nAChRs and their downstream signaling mechanisms.