Abstract
Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.
Highlights
Tein which is able to bind and bunadcletin filaments in The binding of synapsin I to the synaptic vesicle membrane a phosphorylation-dependent fashion
A role of synapsin I phoswith the view that changes in the phosphorylastaiotne phorylation in the regulation of neurotransmitter release is of synapsin I play a functional role in regulating the supported by experiments in which synapsin I was ininteractions between the nerve terminal cytoskeleton jected into the presynaptic digit of the squid giant synapse
Since phosphorylation of synapsin I by CaM kinase I1 markedly altered its effect on actin polymerization triggered by K+ andM$+, we investigated whether asimilar effect was present when actin polymerization was initiated by the addition of dephosphorylated synapsin I, or synapsin I phosphorylated by CaM kinase 11, in theabsence of K+ and M e
Summary
Flavia Valtorta$$, Paul Greengardq, Riccardo Fesce$, Evelina Chieregatti$, and BFeanbfieonatiII. In the present is characterized by high affinity and saturability andis modpaper we have analyzed tehfefects of synapsinI on the ulated by phosphorylation, the binding affinity being reduced kinetics of actin polymerization and their modulation &fold upon phosphorylation of synapsin I by CaM kinase I1 by site-specific phosphorylationofsynapsin I. Theeffect was observed at Greengard, 1987; Petrucci and Morrow, 1987) The bundling both low and high ionicstrength,was specific for syn- activity is decreased after phosphorylation by the catalytic apsin I, and was still present when polymerizationwas subunit of CAMP-dependent protein kinase and virtually triggered by F-actsineeds.Dephosphorylated synapsin abolished after phosphorylation by CaM kinase 11. Phosphorylationby Ca’’/calmodulin-dependent pro- A variety of physiological and pharmacological manipulatein kinase 11 These observations suggest that synap- tions known to stimulate neurotransmitter release have been sin I has a phosphorylation-dependent nucleating ef- shown to increase the stateof phosphorylation of synapsin I fect on actin polymerization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie Blue staining and densitometric scanning of the gels (Ultroscan XL laser densitometer, LKB, Bromma, Sweden)
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