SITE-SPECIFIC PHOSPHORYLATION OF SYNAPSIN-I BY MITOGEN-ACTIVATED PROTEIN-KINASE AND CDK5 AND ITS EFFECTS ON PHYSIOLOGICAL FUNCTIONS

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein k inase II (CaM kinase II), a hitherto unrecognized site (Ser(553)) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo subst rate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed high ly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23 , and glycogen synthase kinase 3 beta, were tested for the in vitro ph osphorylation of synapsin I, Only MAP kinase and Cdk5-p23 phosphorylat ed synapsin I stoichiometrically. The phosphorylation sites were deter mined to be Ser(551) and Ser(553) with Cdk5-p23, and Ser(62), Ser(67), and Ser(551) with MAP kinase, Upon phosphorylation with MAP kinase, s ynapsin I showed reduced F-actin bundling activity, while no significa nt effect on the interaction was observed with the protein phosphoryla ted with Cdk5-p23. These results raise the possibility that the so-cal led proline-directed protein kinases together with CaM kinase II and c AMP-dependent protein kinase play an important role in the regulation of synapsin I function.