Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Abstract

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.Synapsin I is the major synaptic vesicle protein that binds to the Src homology 3 (SH3) domains of the adapter protein Grb2 in vitro (1). This interaction is mediated by domain D of synapsin I, a 23-kDa proline-rich, strongly basic domain located in the COOH-terminal portion of synapsin I (2). It seemed possible that domain D or other proline-rich regions in synapsin I might interact with other SH3 domain-containing proteins within the nerve terminal and that these interactions might have a physiological role in presynaptic function. One such candidate, the SH3 domain-containing nonreceptor tyrosine kinase c-Src, is expressed at high levels in postmitotic neurons and is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity (3-6). Using purified components in vitro, we now report that a domain Dmediated interaction of synapsin I with the SH3 domain of c-Src results in a 5-fold activation of the catalytic activity of c-Src. We also present studies employing a variety of biochemical techniques that serve to further characterize the specificity, subcellular location, regulation, and potential functional consequences of the c-Src͞synapsin I interaction.

A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells

“…Ca 2ϩ influx, which triggers neurotransmitter release, activates src kinase and induces tyrosine phosphorylation of several proteins, including synaptophysin, a synaptic vesicle protein (6)(7)(8). On synaptic vesicles, c-src interacts with synapsin, a perimembrane protein on these vesicles, through its SH3 domain (9,10). These results suggest that src family tyrosine kinases are involved in the regulation of neurotransmitter release; however, no direct evidence to support this has been obtained.…”

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

“…The activation of c-Src in cells expressing GFP-P52 and GFP-P66 demonstrates that c-Src activity can be up-regulated by an adaptor protein. This is not so surprising because several molecules with no intrinsic catalytic activity have been reported to be an activator for c-Src or the Src family tyrosine kinases: it includes hepatitis B virus HBx protein (Klein & Schneider 1997), HIV virus Nef protein (Moare® et al 1997), synapsin I (Onofri et al 1997), b-arrestin 1 (Luttrell et al 1999), and TRAF6 (Wong et al 1999).…”

Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON

“…Synapsins also participate in neurite outgrowth and extension (21,26). These effects are mediated in part by a diverse set of binding proteins such as src (27,28), annexin VI (29), fodrin (30), neurofilaments (31), and actin (32). Synapsins may function as scaffolding proteins that target a diverse set of proteins to presynaptic sites.…”