Specificity of the Binding of Synapsin I to Src Homology 3 Domains

Fabio Benfenati,Paul Greengard,Silvia Giovedı̀,Pietro De Camilli,Flavia Valtorta,Lucilla Bongiorno Borbone,Hung-Teh Kao,Franco Onofri

doi:10.1074/jbc.m006018200

Abstract

Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.

Highlights

Pose modules that regulate a variety of cellular processes by mediating the formation and dissociation of specific intra- and intermolecular protein-protein interactions
In the case of synapsin I, the highest binding levels were found with the Src homology 3 (SH3) domains of c-Src, PI3K, and Grb2; somewhat lower binding levels were found with the SH3 domains of amphiphysin II, PLC␥, amphiphysin I, and N-Grb2; low binding levels were observed with the SH3 domains of p47phox, Crk, and ␣-spectrin, whereas binding was absent with the C-Grb2 SH3 domain
To determine whether a similar SH3 binding pattern was found with endogenous brain proteins and to compare synapsin labeling with the labeling of other abundant SH3-binding proteins such as dynamin I and synaptojanin I, proteins from subcellular fractions obtained from rat forebrain including post-nuclear supernatant of homogenate, crude synaptic vesicles (SV), and highly purified native and synapsin-depleted SV were analyzed for their ability to bind to the various SH3 domains using the overlay assay (Fig. 2)

Summary

Introduction

Pose modules that regulate a variety of cellular processes by mediating the formation and dissociation of specific intra- and intermolecular protein-protein interactions.

Results

Conclusion