PHOSPHORYLATION OF P35

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。这个项目是继前实验室成员德里克麦克拉克林的合作与金勇博士从格林加德的实验室。p35是细胞周期蛋白依赖性激酶5（Cdk 5）激活剂。cdk 5/p35复合物磷酸化在神经系统中具有多功能作用的多种底物。在发育过程中，它参与神经元的分化、迁移、轴突生长和突触发生。P35在体外被发现自磷酸化，这可能表明了一种体内功能调节机制。我们着手确定p35磷酸化位点作为研究其功能的第一步。从昆虫细胞中过量表达和纯化P35，并在体外用cdk 5和32 P-ATP磷酸化。蛋白质经胰蛋白酶消化后，用HPLC分离多肽，用MALDI-TOF、串联MS/MS和HMMS（hypothesis-driven multi-stage MS）鉴定磷酸肽。鉴定出6个磷酸肽，分别对应于两个磷酸化位点。定点突变和P35的胰蛋白酶消化的2D肽图用于确认所鉴定的位点。我们目前正专注于确定一个额外的磷酸化位点所示的磷酸化图像的2D地图从胰蛋白酶消化的磷酸化野生型p35。

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.