RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

See-Ying Tam,Mindy Tsai,Jennifer N Lilla,Janet Kalesnikoff,Ching-Cheng Chen,Alexander G Obukhov

doi:10.1371/journal.pone.0142935

Abstract

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

Highlights

Nerve growth factor (NGF) is a member of the family of neurotrophins which include brain derived growth factor (BDNF) and neurotrophin-3 (NT-3) [1,2]
Using Northern blot and Western blot analyses, we have previously found that RabGEF1 mRNA and protein were expressed in high levels in the brain, suggesting that RabGEF1 may play an important role in the development and function of the central nervous system [25]
We found that all individual lines of PC12 cell transfectants obtained after antibiotic selection and expressing one of the three different RabGEF1-shRNA expression constructs failed to attain confluence even after they were cultured in coated tissue culture plates for a prolonged period of time

Summary

Introduction

Nerve growth factor (NGF) is a member of the family of neurotrophins which include brain derived growth factor (BDNF) and neurotrophin-3 (NT-3) [1,2] These neurotrophins are important for the survival, development, and function of neurons in the central and peripheral nervous systems and they exert their effects through their interactions with specific tyrosine kinase receptors: TrkA (NGF), TrkB (BDNF, NT-3), TrkC (NT-3) [3,4]. NGF-mediated signaling is transmitted retrogradely through axonal transport of signaling endosomes containing NGF, TrkA, and activated signaling intermediate factors such as ERK-kinases [7,8,9] These signaling events result in the induction of neurite outgrowth, a hallmark in PC12 differentiation that is characterized by formation of filamentous actin containing spikes followed by growth and extension of long neurite processes [5]. It has been proposed that the inhibition of Rab activity by TrkA-associated RabGAP5 promotes the diversion of TrkA-containing endocytic vesicles to the formation of signaling endosomes, leading to the propagation of NGF-mediated signaling and neurite outgrowth [12]

Methods

Results

Conclusion