Growth Cone Phosphoproteomics Reveals that GAP-43 Phosphorylated by JNK Is a Marker of Axon Growth and Regeneration

Asami Kawasaki,Masayasu Okada,Atsushi Tamada,Shujiro Okuda,Motohiro Nozumi,Yasuyuki Ito,Daiki Kobayashi,Tokiwa Yamasaki,Ryo Yokoyama,Takeshi Shibata,Hiroshi Nishina,Yutaka Yoshida,Yukihiko Fujii,Kosei Takeuchi,Michihiro Igarashi

doi:10.1016/j.isci.2018.05.019

Abstract

SummaryNeuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

Highlights

The growth cone, a specialized, highly motile structure formed at the tips of extending axons of developing neurons (Dent et al, 2011; Igarashi, 2014), is crucial for accurate synaptogenesis in the developing brain
High Frequency of P-Directed Phosphosites in growth cone membrane (GCM) Phosphoproteomics analysis of GCM fractions isolated from postnatal day 1 (P1) rat forebrain identified more than 30,000 phosphopeptides at greater than 95% confidence
Thresholding with 1% false discovery rate (FDR) extracted 4,596 phosphorylation sites that corresponded to 1,223 proteins

Summary

Introduction

The growth cone, a specialized, highly motile structure formed at the tips of extending axons of developing neurons (Dent et al, 2011; Igarashi, 2014), is crucial for accurate synaptogenesis in the developing brain. To understand the mechanisms underlying neuronal network formation and maintenance, it is essential to elucidate the molecular pathways that determine growth cone behavior. We performed a proteomics analysis of mammalian growth cones and characterized approximately 1,000 unique proteins (Nozumi et al, 2009; see Estrada-Bernal et al, 2012). The results of this analysis revealed novel molecular mechanisms underlying nerve growth (Igarashi, 2014; Nozumi et al, 2017; Honda et al, 2017a, 2017b)

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Discussion

Conclusion