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J. Biol. Chem., Vol. 282, Issue 21, 15754-15767, May 25, 2007

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Synapsin Phosphorylation by Src Tyrosine Kinase Enhances Src Activity in Synaptic Vesicles^*

Franco Onofri, Mirko Messa, Vittoria Matafora^¶, Giambattista Bonanno, Anna Corradi, Angela Bachi^¶, Flavia Valtorta^||, and Fabio Benfenati¹

From the Department of Experimental Medicine, University of Genova, 16132 Genova, the Department of Neuroscience and Brain Technologies, The Italian Institute of Technology Central Laboratories, 16163 Genova, the ^¶San Raffaele Scientific Institute, 20132 Milano, and the ^||San Raffaele Vita-Salute University and The Italian Institute of Technology, Unit of Molecular Neuroscience, 20132 Milano, Italy

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr³⁰¹, which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca²⁺-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Received for publication, February 5, 2007 , and in revised form, March 21, 2007.

^* This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (to F. B.), the Fisher Center for Alzheimer's Disease Research (to F. B.), the Cariplo Foundation (to F. B. and F. V.), and Italian Ministry of Research Grants FIRB 2001, PRIN 2004, PRIN 2005, and PRIN 2006 (to F. B., F. V., and F. O.), and Telethon-Italy Grant GGP05134 (to F. B. and F. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Via Benedetto XV n. 3, 16132 Genova, Italy. Tel.: 39-010-3538183; Fax: 39-010-3538194; E-mail: benfenat{at}unige.it.

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