Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.
Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Mahdokht
0000-0002-3898-5197
M
Kohansal-Nodehi
Interactomics and Intracellular Trafficking laboratory, National University of Singapore, Singapore, Singapore.|Department of Physiology, National University of Singapore, Singapore, Singapore.|Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|Neurobiology/Ageing Programme, National University of Singapore, Singapore, Singapore.
John Je
0000-0002-5615-1014
JJ
Chua
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.|Bioanalytics Group, University Medical Center Göttingen, Göttingen, Germany.
Henning
H
Urlaub
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Reinhard
0000-0003-1542-3498
R
Jahn
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Dominika
0000-0001-7911-0867
D
Czernik
D000818:Animals; D001921:Brain / Q000502:physiology; D005089:Exocytosis; D009474:Neurons / Q000502:physiology*; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000032:analysis; D017208:Rats, Wistar
D011506:Proteins; D020543:Proteome
active zone; exocytosis; neuroscience; phosphoproteomics; phosphorylation; protein kinase; rat; synaptosome