Tropomyosin-related kinase A (TrkA) is the cognate receptor for the prototypic neurotrophin nerve growth factor (NGF), which is a well-known member of the neurotrophin family. Binding of NGF to TrkA receptors induces internalization and trafficking of NGF/TrkA complex through the endocytic pathway. Previous study in our lab found a novel role of endophilin B1, which belongs to a family of lipid binding proteins that mediates generation of high curvature membranes. It is reported that knock-down of endophilin B1 triggers precocious targeting of NGF/TrkA to late endosomes and lysosomes. This leads to marked reduction in TrkA level and attenuation of NGF-induced gene transcription and neurite outgrowth. Recently, our lab has also demonstrated endophilin B1 as a Cdk5 substrate and it is pho...[ Read more ]

Tropomyosin-related kinase A (TrkA) is the cognate receptor for the prototypic neurotrophin nerve growth factor (NGF), which is a well-known member of the neurotrophin family. Binding of NGF to TrkA receptors induces internalization and trafficking of NGF/TrkA complex through the endocytic pathway. Previous study in our lab found a novel role of endophilin B1, which belongs to a family of lipid binding proteins that mediates generation of high curvature membranes. It is reported that knock-down of endophilin B1 triggers precocious targeting of NGF/TrkA to late endosomes and lysosomes. This leads to marked reduction in TrkA level and attenuation of NGF-induced gene transcription and neurite outgrowth. Recently, our lab has also demonstrated endophilin B1 as a Cdk5 substrate and it is phosphorylated by Cdk5 at Thr145. However, whether Cdk5 may affect NGF/TrkA trafficking through phosphorylation of endophilin B1, thereby regulating NGF-mediated downstream functions, is still an enigma. In this study, we established a cell-based system using streptavidin-fluor568 conjugated dye to label biotinylated TrkA in AP-TrkA overexpressing primary cultured cortical neurons, to examine TrkA trafficking induced by NGF treatment. We further examined the importance of Cdk5 in the regulation of TrkA trafficking by endophilin B1. Cdk5 activity was reduced by treatment with Cdk5 inhibitor roscovitine or RNA interference in cultured cortical neurons. Both roscovitine treatment and knock-down of Cdk5 affected trafficking of TrkA, possibly by blocking receptor internalization or enhancing the early sorting of receptors from early endosome to recycling endosome. We then examined the significance of Thr145 phosphorylation of endophilin B1 on the trafficking of TrkA. We found that phosphorylation of endophilin B1 by Cdk5 at Thr145 may not be implicated in the regulation of TrkA trafficking by Cdk5. These observations suggest that while Cdk5 activity may have an impact on the regulation of NGF/TrkA trafficking, its effect is possibly independent from its phosphorylation of endophilin B1. Our data call for further investigations to delineate the precise mechanisms implicated.