Roles of MAP Kinase-dependent Phosphorylation of Synapsin I in the Regulation of Neurotransmitter Release

我们一直在使用LC/MS系统研究大脑特定的磷蛋白，在该系统中，毛细管液相色谱柱与电喷雾质谱仪在线连接。本研究旨在研究突触素I、GAP-43和MAP1B等脑特异性磷酸蛋白的翻译后修饰，这些蛋白参与突触的延伸和突触的形成，其功能受包括MAP和PKC在内的蛋白激酶级联网络的调节，我们可以利用纳米螺旋电离方法建立一种超灵敏的体内磷酸化检测方法。这些结果揭示了包括Ser(Thr)-Pro基序在内的新的磷酸化位点，这表明这些蛋白是在体内高表达的所谓的脯氨酸导向的蛋白激酶的底物，如MAP激酶和CDK5。因此，这些具有SP特异性的蛋白激酶直接参与神经递质释放和突触形成的调节。我们还发现MAP 1B的磷酸化影响其与微管的结合。磷酸化部位位于微管蛋白结合部位附近。这些结果表明，在突触发生过程中，MAP1B功能的调节中可能存在着与Pro有关的蛋白激酶，如MAP1B。

We have been studying brain-specific phosphoproteins using an LC/MS system, in which a capillary HPLC column is connected on-line to an electrospray mass spectrometer. In this research, our aim is to study posttranslational modifications of brain-specific phosphoproteins such as synapsin I, GAP-43, and MAP1B.These proteins are involved in the neurite extension and synapse formation, and their functions are regulated by the network of protein kinase cascades including MAP kinase and PKC, We could establish an ultra-sensitive assay for in vivo phosphorylation using nanospray ionization method. The results obtained revealed novel phosphorylation sites including Ser (Thr)-Pro motif, suggesting that these proteins are in vivo substrates of so-called proline-directed protein kinase such as MAP kinase and Cdk5, which are highly expressed in the brain. These protein kinases with the SP specificity, therefore, are involved directly in the regulation of neurotransmitter release and synapse formation. We also found that phosphorylation of MAP 1B affected its binding to microtubules. The phosphorylation sites are found near the tubulin-binding site. These results suggest that the proline-directed protein kinases such as MAP kinase play important roles in the regulation of MAP1B function during synaptogenesis.