title: Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation.
abstract: The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.
authors.affiliation: Center of Neuropharmacology, Institute of Pharmacological Sciences, University of Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Verona
authors.forename: S
authors.initials: S
authors.lastname: Zanotti
authors.forename: T
authors.initials: T
authors.lastname: Schäfer
authors.forename: G
authors.initials: G
authors.lastname: Racagni
authors.forename: M
authors.initials: M
authors.lastname: Popoli
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000378:metabolism; D050857:Synaptotagmins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
chemical_list: D000954:Antigens, Surface; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D005982:Glutathione Transferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D002118:Calcium