A synaptic vesicle-associated form of CaM kinase II was identified as a major binding protein for the C-terminal region of synapsin I. The enzyme can regulate vesicle- and actin-binding activity of synapsin I by phosphorylation. The presence of a preassembled complex of CaM kinase II and synapsin I at the vesicle membrane may mediate sustained Ca2+-independent phosphorylation of synapsin I beyond the termination of the Ca2+ signal. The mechanism responsible for the targeting of CaM kinase II to the synaptic vesicle membrane is unknown. The vesicle-associated form might represent a novel gene product (e.g., the product of a previously unidentified gene, or a new isoform derived by alternative splicing of one of the four identified subunits) that contains a structural domain that is responsible for membrane targeting. Alternatively, it might be identical in primary structure to the soluble form, and is targeted to the membrane by a posttranslational lipid modification or via an interaction with a specific """"""""receptor"""""""" protein on the synaptic vesicle membrane. To distinguish between these possibilities, purified soluble kinase and vesicle-associated kinase will be subjected to proteolytic/chemical fragmentation, followed by analysis using mass spectroscopy. Determining the exact mass of derived peptides of both proteins will allow us to detect changes in either the primary sequence or a posttranslational modification. In preliminary studies with Dr. Chait, several novel phosphorylation sites on CaM kinase 11 have also been revealed. Further studies will be performed to establish the identity of these sites.
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