PHOSPHORYLATION OF P35

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目是前实验室成员 Derek McLachlin 与 Greengard 博士实验室的 Yong Kim 合作开展的。 p35 是一种细胞周期蛋白依赖性激酶 5 (Cdk5) 激活剂。 Cdk5/p35 复合物磷酸化多种底物，这些底物在神经系统中具有多功能作用。在发育过程中，它参与神经元分化、迁移、轴突生长和突触发生。在体外发现 P35 自磷酸化，这可能表明体内的功能调节机制。我们着手鉴定 p35 磷酸化位点，作为研究其功能的第一步。 P35 从昆虫细胞中过表达和纯化，并在体外用 cdk5 和 32P-ATP 磷酸化。对蛋白质进行胰蛋白酶消化，并通过 HPLC 分离肽，通过 MALDI-TOF、随后串联 MS/MS 以及 HMMS（假设驱动的多级 MS）鉴定磷酸肽。鉴定出六种磷酸肽，它们对应于两个磷酸化位点。使用定点诱变和 P35 胰蛋白酶消化的 2D 肽图来确认所识别的位点。我们目前的重点是确定磷酸化野生型 p35 胰蛋白酶消化的 2D 图谱磷酸化图像显示的额外磷酸化位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is following a former lab member Derek McLachlin¿s collaboration with Yong Kim from Dr. Greengard's lab. p35 is a cyclin-dependent kinase 5 (Cdk5) activator. Cdk5/p35 complex phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. P35 was found autophosphorylated in vitro, which may indicate a mechanism of functional regulation in vivo. We set out to identify the p35 phosphorylation sites as an initial step towards studying its function. P35 were over-expressed and purified from insect cells and phosphorylated with cdk5 and 32P-ATP in vitro. Protein was tryptic digested and peptides was separated by HPLC, phosphopeptide were identified by MALDI-TOF followed by tandem MS/MS, as well as HMMS (hypothesis-driven multi-stage MS). Six phosphopeptides were identified which correspondent to two phosphorylation sites. Site directed mutagenesis and 2D peptide map of the tryptic digest of P35 were used to confirm the identified sites. We are currently focusing on identifying an additional phosphorylation site shown by the phospho-image of the 2D map from tryptic digest of phosphorylated wild type p35.