Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

Richard D Smith,Yingchun Wang,Marina A Gritsenko,Daniel J Orton,Matthew E Monore,Jon M Jacobs,Xiahe Huang,Feng Yang,Richard L Klemke,Rui Zhao,Wei Wang,Olivier C Pertz,David G Camp,Xinning Jiang,Samuel O Purvine,Yi Fu

doi:10.1074/jbc.m111.236133

Abstract

Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

Highlights

From the ‡Department of Pathology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, the ¶Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, and the §State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
Because the cell body remains on the surface of the filter and neurite growth occurs exclusively through the pore to the lower surface, it is possible to selectively purify this structure from the polarized neuron for spatial proteomic analysis
Akt activity was not inhibited by LPA-induced neurite retraction (Fig. 4C). These findings demonstrate that in polarized neurons MEK/ ERK kinase activity is highly compartmentalized within the neurite and that during neurite collapse MEK/ERK activity is shut off

Summary

Introduction

LPA-mediated neurite collapse induced a rapid decrease in ERK and MEK activities, which correlated with neurite retraction kinetics (Fig. 4, A and B). These findings suggest that LPA induces neurite collapse and ERK activity by interfering with integrin adhesion signaling to Rac/Cdc42/PAK and MEK-Ser-298 phosphorylation. In support of these findings, LPA blocked ERK activation and MEK Ser-298 phosphorylation induced by integrin-mediated NIE-115 cell attachment and spreading on laminin (Fig. 4G).

Results

Conclusion