Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase

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... s, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2. Neurotransmitter secretion occurs via a tightly regulated membrane trafficking cycle localized to the presynaptic terminal. Many stages of this cycle, such as the targeting and docking of vesicles at active zone membranes, vesicle fusion, and endocytosis, are mediated by the formation of protein complexes (reviewed in Refs. 1 and 2). Protein phosphorylation, a ubiquitous mechanism of cellular regulation, plays an important role in the modulation of the synaptic vesicle cycle. Multiple examples of phosphorylation-mediated regulation of protein binding in the synapse have been reported. One well characterized example is the phosphorylation-dependent association of the actin-binding protein synapsin with synaptic vesicles, an interaction implicated in the maintenance of a reserve pool of vesicles (3, 4). Likewise, proteins involved in the docking and fusion of vesicles, termed SNAREs, 1 are sub-strates of several protein kinases, and phosphorylation has been reported to alter their binding to other proteins and to each other (5-8). Synaptic vesicle protein 2 (SV2), a protein common to all neurons (9, 10), has been implicated in the regulation of synaptic vesicle exocytosis (11, 12) . Three separately encoded isoforms, termed SV2A, SV2B, and SV2C, have been identified (13-18). Loss of the most widely expressed isoform, SV2A, results in aberrant neurotransmission and death, indicating that SV2 is an essential protein (11, 12). We previously reported that SV2A interacts with the synaptic vesicle protein synaptotagmin, a calcium-binding protein that is necessary for normal calcium-stimulated neurotransmission (19). The SV2synaptotagmin interaction is modulated by calcium, suggesting that it plays a role in the regulation of exocytosis. The amino terminus of SV2, which mediates its interaction with synaptotagmin, contains substrate consensus sites of several protein kinases. In addition, SV2 was previously reported to be phosphorylated in vitro under conditions permissive to casein kinase I activity (20). In order to determine whether SV2 is phosphorylated in vivo and whether phosphorylation regulates its action, we examined SV2 phosphorylation in intact nerve terminals and in synaptic vesicles. We report that SV2 is a phosphoprotein in the synapse, that it is phosphorylated on its amino terminus, and that phosphorylation increases the binding of SV2 to synaptotagmin. These results suggest that the SV2-synaptotagmin interaction is regulated by changes in SV2 phosphorylation levels. EXPERIMENTAL PROCEDURES Materials-Radiolabeled orthophosphate and adenosine triphosphate were purchased from NEN Life Science Products, protein A-Sepharose was from Amersham Pharmacia Biotech, glutathione-agarose was from Sigma, and recombinant rat casein kinase I was from Calbiochem. Sequencing grade trypsin was purchased from Promega (Madison, WI), and Ni 2ϩ -NTA-agarose was from Qiagen. cAMP-dependent protein kinase was kindly provided by Dr. Brian Murphy (University of Washington, Seattle, WA). Synaptosome and Synaptic Vesicle Isolation-Crude synaptosomes and synaptic vesicles were prepared as described by Huttner et al. (21). Briefly, fresh rat brains were homogenized in buffered sucrose (10 mM Hepes, pH 7.5, 0.3 M sucrose) with 10 strokes in a glass-Teflon homogenizer (0.004 -0.006-inch clearance). Homogenates were centrifuged at 1000 ϫ g to remove nuclei and intact cells. Crude synaptosomes were recovered from the remaining supernatant by centrifugation at 25,000 ϫ g for 13 min. The protein concentration of resuspended synaptosomes was determined with the Bio-Rad protein assay using bovine serum albumin as a standard. To obtain a crude synaptic vesicle preparation, synaptosomes were washed, resuspended in HBS (142 mM NaCl, 2.4 mM KCl, 1 mM MgCl 2 , 0.1 mM EGTA, 10 mM Hepes, pH 7.5, 5 mM D-glucose), and then hypotonically lysed by diluting 1:9 with water, rehomogenizing, and incubating for 30 min on ice. Heavy membranes