Identification of Peripherin as a Akt Substrate in Neurons

Hiroyuki Konishi,Kazuhiko Namikawa,Keiji Shikata,Yuji Kobatake,Taro Tachibana,Hiroshi Kiyama

doi:10.1074/jbc.m611703200

Abstract

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.

Highlights

Neuronal cell bodies, Schwann cells secrete trophic factors [16]
We demonstrate that peripherin, which is a peripheral nervous system neuron-specific intermediate filament protein, is a novel Akt substrate, and that Ser66 of peripherin is the phosphorylation site
Peripherin phosphorylation is apparently induced in motor neurons after nerve injury, suggesting that the Akt-mediated peripherin phosphorylation may play a role in motor nerve regeneration

Summary

Introduction

Neuronal cell bodies, Schwann cells secrete trophic factors [16] Such factors released from those glial cells include a wide range of growth factors such as nerve growth factor, brainderived neurotrophic factor, glial cell line-derived neurotrophic factor, and fibroblast growth factor-2 [17,18,19]. They are known to activate the phosphatidylinositol 3-kinase-Akt pathway in injured neurons via their respective receptors (20 –22). Peripherin phosphorylation is apparently induced in motor neurons after nerve injury, suggesting that the Akt-mediated peripherin phosphorylation may play a role in motor nerve regeneration

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