Roles of C Kinase Substrate Protein, MARCKS and GAP-43, in Neurotransmitter Release

我们建立了一个LC/MS系统，将毛细管柱在线连接到电喷雾质谱仪上，并研究了大脑特异的磷酸蛋白如Marcks、GAP-43、Synapsin I和MAP1B的翻译后修饰。结果发现了包括Ser(Thr)-Pro基序在内的新的磷酸化位点，表明这些PKC的底物蛋白，钙调蛋白依赖的蛋白激酶II或酪蛋白激酶II，是所谓的脯氨酸导向的蛋白激酶的体内底物。对在脑中高表达的MAP和CDK5进行的体外磷酸化实验表明，这些蛋白是定点磷酸化的。脑内也可能存在其他具有SP特异性的未知蛋白激酶。突触素I的磷酸化影响肌动蛋白细丝的结合活性。这些结果表明，除了PKC和钙调蛋白依赖的蛋白激酶II外，Pro导向的蛋白激酶II在神经递质释放的调节中起着重要作用。此外，我们还证明了MAP1B的磷酸化对于培养的皮质神经元中突触的形成是必不可少的。这些研究证实了脯氨酸导向的蛋白激酶在大脑功能中的重要性。

We have established an LC/MS system, in which a capillary HPLC column is connected on-line to an electrospray mass spectrometer, and studied posttranslational modificaitons of brain-specific phosphoproteins such as MARCKS,GAP-43, synapsin I,and MAP1B.The results obtained revealed novel phosphorylation sites including Ser (Thr) -Pro motif, suggesting that these substrate proteins of PKC,calmodulin-dependent protein kinase II or casein kinase II,are in vivo substrates of so-called proline-directed protein kinase. The in vitro phosphorylation experiments with MAP kinase and Cdk5, which are highly expressed in the brain, demonstrated that these kinases site-specifically phosphorylate the proteins. Other unknown protein kinases with the SP specificity, may also exist in the brain. Phosphorylation of synapsin I affected the bunding activity of actin filaments. These results suggest that the proline-directed protein kinases in addition to PKC and calmodulin-dependent protein kinase II play important roles in the regulation of neurotransmitter release. Furthermore, we have also demonstrated that the phosphorylation of MAP1B is essential for the synapse formation in the cultured cortical neurons. These studies established the importance of the proline-directed protein kinases in the brain funcitons.