RESUMO
Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Semaforina-3A/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Axônios/patologia , Células Cultivadas , Quinase 5 Dependente de Ciclina/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Semaforina-3A/genética , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Slo2 channels are a type of sodium-activated K+ channels and possess a typical PDZ binding motif at the carboxy-terminal end. Thus, we investigated whether Slo2 channels bind to PSD-95, because it is well known that other types of K+ channels, voltage-gated and inward rectifier K+ channels, bind to PSD-95 via the PDZ binding motif and are involved in excitatory synaptic transmission. By using an extract prepared from cultured neocortical neurons, we demonstrated a biochemical interaction between mSlo2 channels and PSD-95, and a mutational analysis revealed that mSlo2 channels bound to the first PDZ domain of PSD-95 via the PDZ binding motif. To investigate the expression of mSlo2 protein in primary neocortical neurons, we raised anti-mSlo2 channel antibody and immunostained neocortical neurons. The immunocytochemical study showed that mSlo2 channels partly colocalized with PSD-95 in mouse neocortical neurons.
