Protein phosphorylation in depolarized synaptosomes: Dissecting primary effects of calcium from synaptic vesicle cycling

TY - JOUR

T1 - Protein phosphorylation in depolarized synaptosomes: Dissecting primary effects of calcium from synaptic vesicle cycling

AU - Silbern, Ivan

AU - Pan, Kuan-Ting

AU - Fiosins, Maksims

AU - Bonn, Stefan

AU - Rizzoli, Silvio O

AU - Fornasiero, Eugenio F

AU - Urlaub, Henning

AU - Jahn, Reinhard

N1 - Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2021/2/12

Y1 - 2021/2/12

N2 - Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons.

AB - Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons.

U2 - 10.1016/j.mcpro.2021.100061

DO - 10.1016/j.mcpro.2021.100061

M3 - SCORING: Journal article

C2 - 33582301

VL - 20

JO - MOL CELL PROTEOMICS

JF - MOL CELL PROTEOMICS

SN - 1535-9476

M1 - 100061

ER -