The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation

PLoS Biol. 2019 Mar 1;17(3):e3000170. doi: 10.1371/journal.pbio.3000170. eCollection 2019 Mar.

Abstract

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism
  • Calmodulin / metabolism
  • Cyclin-Dependent Kinase 5 / metabolism
  • Male
  • Mass Spectrometry
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Potassium Chloride / pharmacology
  • Presynaptic Terminals / metabolism*
  • Presynaptic Terminals / physiology
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction / physiology
  • Synaptosomes / metabolism*
  • Synaptosomes / physiology

Substances

  • Calmodulin
  • Phosphoproteins
  • Potassium Chloride
  • Cyclin-Dependent Kinase 5
  • Calcium

Grants and funding

KEK was funded by the Lundbeck Foundation (R83-2011-8143), The Danish Council for Independent Research and FP7 Marie Curie Actions – COFUND (DFF – 1325-00154), and the Carlsberg Foundation (CF15-1056 and CF16-0066). Additional funding was from Christian og Ottilia Brorsons Rejselegat, Beckett-fonden, Etly og Jørgen Stjerngrens Fond, Dagmar Marshalls Fond, Grosserer L. F. Foghts Fond, and Brødrene Hartmanns Fond. MEG was funded by the Rebecca L Cooper Medical Research Foundation. MEG and PJR were funded by National Health and Medical Research Project Grants (1079160, 1077989, 1052494, and 571070). Additional funding was by the Brain Foundation, Australian Cancer Research Foundation, Cancer Institute New South Wales, Zero Childhood Cancer, Ramaciotti Foundation, Honda Foundation, and Bruce Wall Estate. SS and DD were funded by the German Research Council (DFG, SFB1089, SPP1757, SCHO 820/6-1, SCHO 820/4-1, DI853/3-2, DI853/7-1) and local funding (BONFOR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.