Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

Abstract

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.DOI: http://dx.doi.org/10.7554/eLife.14530.001

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

“…The CNome suggests expanded targets for CN both pre-and post-synaptically and points particularly to unexplored roles for CN in the presynaptic active zone, which coordinates neurotransmitter release ( Figure 7D). Identified SLiMs in BSN, PCLO, RIMS1, and RIMS2 suggest that these proteins, whose phosphorylation decreases during exocytosis, are regulated directly by CN (Kohansal-Nodehi et al, 2016). In sum, the network presented here provides a road map for targeted elucidation of CN signaling pathways in the heart, brain and other tissues.…”

Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin

“…At the presynaptic site, CaMKII phosphorylates a variety of proteins, including the synaptic vesicle proteins synapsin I, syntaxin 1A, synaptotagmin I as well as Ca v 1 L-type calcium channels, thereby modulating synaptic vesicle trafficking and exocytosis ( Llinás et al, 1991 ; Fukunaga et al, 1995 ; Hilfiker et al, 1999 ; Ohyama et al, 2002 ; Abiria and Colbran, 2010 ; Jenkins et al, 2010 ). However, a recent proteomics study on synaptosomes uncovered no phosphorylation site to be induced by depolarization and Ca 2+ entry within a C 2 domain ( Kohansal-Nodehi et al, 2016 ). Concordantly, reports about biochemical regulations of C 2 domains are rare, e.g., the non-Ca 2+ binding C 2 domain of a novel PKC from Aplysia was reported to display higher phospholipid affinity upon phosphorylation ( Pepio and Sossin, 2001 ).…”

Distinct functions of a cGMP-dependent protein kinase in nerve terminal growth and synaptic vesicle cycling

“…Presynaptic Ca 2+ (Hosoi et al, 2009), and Synaptotagmin (Syt) (Poskanzer et al, 2003;Yao et al, 2012) and SNARE (Deák et al, 2004;Xu et al, 2013;Zhang et al, 2013) proteins are thought to be involved in coupling SV exocytosis and endocytosis. Many presynaptic proteins that regulate SV exocytosis and endocytosis undergo phosphorylation/dephosphorylation (Turner et al, 1999;Clayton et al, 2007;Kohansal-Nodehi et al, 2016). Changes in phosphorylation state can alter the affinity of proteins for some of their binding partners (Clayton et al, 2009;Cho et al, 2015;Geng et al, 2016), as well as alter the probability of ion channels opening (Huang and Zamponi, 2017).…”