Graham Mark E, Anggono Victor, Bache Nicolai, Larsen Martin R, Craft George E, Robinson Phillip J
Cell Signaling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.
J Biol Chem. 2007 May 18;282(20):14695-707. doi: 10.1074/jbc.M609713200. Epub 2007 Mar 20.
Dynamin I (dynI) is phosphorylated in synaptosomes at Ser(774) and Ser(778) by cyclin-dependent kinase 5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis, and in PC12 cells on Ser(857). Hierarchical phosphorylation of Ser(774) precedes phosphorylation of Ser(778). In contrast, Thr(780) phosphorylation by cdk5 has been reported as the sole site (Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation was not detectable. Mutation of Ser(774), Ser(778), and Thr(780) confirmed that Thr(780) phosphorylation is restricted to in vitro conditions. Mass spectrometry of (32)P-labeled phosphopeptides separated by two-dimensional mapping revealed seven in vivo phosphorylation sites: Ser(774), Ser(778), Ser(822), Ser(851), Ser(857), Ser(512), and Ser(347). Quantification of (32)P radiation in each phosphopeptide showed that Ser(774) and Ser(778) were the major sites (up to 69% of the total), followed by Ser(851) and Ser(857) (12%), and Ser(853) (2%). Phosphorylation of Ser(851) and Ser(857) was restricted to the long tail splice variant dynIxa and was not hierarchical. Co-purified, (32)P-labeled dynIII was phosphorylated at Ser(759), Ser(763), and Ser(853). Ser(853) is homologous to Ser(851) in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results suggest a unique role for the long splice variants of dynI and dynIII in nerve terminals.
动力蛋白I（dynI）在突触小体中，其Ser（774）和Ser（778）位点被细胞周期蛋白依赖性激酶5磷酸化，以调节发动蛋白结合蛋白I的募集，从而参与突触小泡的内吞作用；在PC12细胞中，dynI的Ser（857）位点发生磷酸化。Ser（774）的分级磷酸化先于Ser（778）的磷酸化。相比之下，有报道称cdk5介导的Thr（780）磷酸化是唯一的磷酸化位点（富泽 圭、砂田幸司、卢逸峰、小田阳、衣努田真、大岛敏、齐藤敏、魏方宇、松下正、李世堂、筒井健、久永正司、三木茂、武井博、松井博史，《细胞生物学杂志》，2003年，第163卷，第813 - 824页）。为了解决这一差异并更好地理解dynI磷酸化的生物学作用，我们对大鼠脑神经末梢dynI的所有磷酸化位点进行了系统鉴定。通过磷酸氨基酸分析，仅发现了磷酸丝氨酸残基。未检测到Thr（780）的磷酸化。Ser（774）、Ser（778）和Thr（780）的突变证实，Thr（780）的磷酸化仅限于体外条件。通过二维图谱分离的（32）P标记磷酸肽的质谱分析揭示了7个体内磷酸化位点：Ser（774）、Ser（778）、Ser（822）、Ser（851）、Ser（857）、Ser（512）和Ser（347）。对每个磷酸肽中（32）P辐射的定量分析表明，Ser（774）和Ser（778）是主要位点（占总量的69%），其次是Ser（851）和Ser（857）（12%），以及Ser（853）（2%）。Ser（851）和Ser（857）的磷酸化仅限于长尾剪接变体dynIxa，且不存在分级现象。共纯化的（32）P标记的动力蛋白III在Ser（759）、Ser（763）和Ser（853）位点发生磷酸化。Ser（853）与dynIxa中的Ser（851）同源。这些结果确定了dynI中的所有主要和几个次要磷酸化位点，并首次测量了它们的相对丰度以及对去极化的相对反应。多个磷酸化位点表明新的蛋白激酶和新的蛋白质 - 蛋白质相互作用对突触小泡内吞作用进行了精细调节。dynI和dynIII同源位点的磷酸化表明它们在机制上具有高度相似性。结果表明dynI和dynIII的长剪接变体在神经末梢中具有独特作用。
