PHOSPHORYLATION OF P35

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目是关于前实验室成员德里克·麦克拉奇林？S与格林加德博士实验室的金勇合作的。P35是一种细胞周期蛋白依赖性激酶5(CDK5)激活剂。CDK5/p35复合体使多种底物磷酸化，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、轴突生长和突触形成。P35在体外被发现自磷酸化，这可能是体内功能调节的机制之一。我们开始确定p35的磷酸化位点，作为研究其功能的第一步。从昆虫细胞中高效表达和纯化P35，并在体外用CDK5和32P-ATP进行磷酸化。蛋白质经胰酶消化后，用高效液相色谱分离多肽，用MALDI-TOF和串联MS/MS以及假说驱动的多阶段MS鉴定磷酸多肽。共鉴定出6个磷酸化多肽，分别对应于两个磷酸化位点。利用定点突变和P35胰酶二维肽图谱对所鉴定的位点进行鉴定。我们目前正专注于从磷酸化的野生型p35的胰酶消化中鉴定2D MAP的磷酸图像所示的另一个磷酸化位点。

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.