PHOSPHORYLATION OF P35

P35 的磷酸化

• 批准号: 8169118
• 负责人: YONG I KIM
• 金额: $ 0.12万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169118
• 关键词: Axon; Cells; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyclin-Dependent Kinase 5; Development; Funding; Grant; High Pressure Liquid Chromatography; Image; In Vitro; Insecta; Institution; Maps; Nervous system structure; Neuronal Differentiation; Peptide Mapping; Peptides; Phosphopeptides; Phosphorylation; Phosphorylation Site; Proteins; Regulation; Research; Research Personnel; Resources; Role; Site; Site-Directed Mutagenesis; Source; Staging; United States National Institutes of Health; in vivo; member; migration; synaptogenesis

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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 该项目正在遵循前实验室成员德里克·麦克拉克林（Derek McLachlin）与Greengard博士实验室的Yong Kim的合作。 p35是细胞周期蛋白依赖性激酶5（CDK5）活化剂。 CDK5/p35复合物磷酸化在神经系统中具有多功能作用的潜水员底物。在开发过程中，它参与神经元分化，迁移，轴突产物和突触发生。发现p35在体外被自磷酸化，这可能表明体内功能性调节的机制。我们着手将p35磷酸化位点确定为研究其功能的第一步。 p35过表达并从昆虫细胞中纯化，并在体外用CDK5和32P-ATP磷酸化。胰蛋白酶消化并通过HPLC分离肽，通过MALDI-TOF鉴定磷酸肽，然后通过串联MS/MS以及HMM（假设驱动的多阶段MS）鉴定。鉴定出六个对应于两个磷酸化位点的磷酸肽。 p35的胰蛋白酶消化物的定向诱变和2D胡椒图用于确认已识别的位点。我们目前正在专注于鉴定来自磷酸化野生型p35的2D图的磷酸化图像所示的附加磷酸化位点。