The in vivo phosphorylation sites of rat brain dynamin I
The in vivo phosphorylation sites of rat brain dynamin I
复制标题
DOI：
10.1074/jbc.m609713200
复制
发表时间：
2007-05-18
影响因子：
4.8
通讯作者：
Robinson, Phillip J.
中科院分区：
文献类型：
--
作者：
Graham, Mark E.;Anggono, Victor;Robinson, Phillip J.
动力蛋白I(DynI)在Ser(774)和Ser(778)的突触体中被细胞周期蛋白依赖的激酶5磷酸化，以调节突触囊泡内吞的Syndapin I的募集，并在PC12细胞的Ser(857)上被磷酸化。丝氨酸(774)的分级磷酸化先于丝氨酸(778)的磷酸化。相反，CDK5的Thr(780)磷酸化已被报道为唯一的位点(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.和Matsui，H.(2003)J.Cell Biol)。163,813-824)。为了解决这一差异，并更好地了解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的鉴定。用磷酸氨基酸分析，发现只有磷酸丝氨酸残基。未检测到苏氨酸受体(780)的磷酸化。Ser(774)、Ser(778)和Thr(780)的突变证实了Thr(780)的磷酸化仅限于体外条件。经双向图谱分离的P-32标记磷酸肽的质谱图显示，体内有7个磷酸化位点：Ser(774)、Ser(778)、Ser(822)、Ser(851)、Ser(857)、Ser(512)和Ser(347)。各磷酸肽的P-32辐射定量显示，Ser(774)和Ser(778)是主要的结合部位(占总数的69%)，其次是Ser(851)和Ser(857)(12%)，Ser(853)(2%)。Ser(851)和Ser(857)的磷酸化仅限于长尾剪接变异体dynIxa，没有层次性。共纯化的P-32标记的dynIII在Ser(759)、Ser(763)和Ser(853)处被磷酸化。在dynIxa中，Ser(853)与Ser(851)同源。这些结果确定了dynI中所有的主要和几个次要的磷酸化位点，并提供了它们的相对丰度和对去极化的相对反应的第一个度量。多个磷酸化位点暗示了新的蛋白激酶和新的蛋白-蛋白相互作用对突触囊泡内吞作用的微妙调节。同源的dynI和dynIII的磷酸化表明有很高的机制相似性。结果表明，dynI和dynIII的长剪接变体在神经末梢中具有独特的作用。
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 P-32-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 P-32 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, P-32- 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.