Krieger Jonathan R, Taylor Paul, Moran Michael F, McGlade C Jane
Program in Cell Biology, The Hospital For Sick Children, Toronto, ON, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada.
Proteomics. 2015 Jan;15(2-3):434-46. doi: 10.1002/pmic.201400232.
Numb is an adaptor protein that functions in the endocytosis and intracellular trafficking of membrane receptors and adhesion molecules. Previous studies have indicated that Numb localization and function are regulated through phosphorylation by atypical protein kinase C at several key sites. Here, using LC-MS/MS, we report the identification of 25 serine/threonine Numb phosphorylation sites, and a single tyrosine phosphorylation site. Amino acid sequences flanking several of the sites identified conform to consensus motifs for cyclin-dependent kinase 5 (CDK5). In vitro kinase assays and immunoblotting confirmed that CDK5 phosphorylates Numb. LC-MS/MS analysis identified specific CDK5-directed phosphorylation of Numb at position S288 and at two additional regions. Therefore, Numb is likely to exist in multiple phospho-isoforms, and may be subject to phosphorylation-mediated regulation downstream of CDK5. These findings provide a basis for further investigations into the complex role of Numb phosphorylation in regulating its subcellular localization, protein interactions, and function. All MS data have been deposited in the ProteomeXchange with identifier PXD000997 (http://proteomecentral.proteomexchange.org/dataset/PXD000997).
Numb是一种衔接蛋白，在膜受体和黏附分子的内吞作用及细胞内运输过程中发挥作用。先前的研究表明，Numb的定位和功能通过非典型蛋白激酶C在几个关键位点的磷酸化作用来调节。在此，我们使用液相色谱-串联质谱法（LC-MS/MS）报告了25个丝氨酸/苏氨酸Numb磷酸化位点以及一个酪氨酸磷酸化位点的鉴定结果。所鉴定的几个位点两侧的氨基酸序列符合细胞周期蛋白依赖性激酶5（CDK5）的共有基序。体外激酶测定和免疫印迹证实CDK5使Numb磷酸化。LC-MS/MS分析确定了CDK5在S288位点及另外两个区域对Numb的特异性磷酸化作用。因此，Numb可能以多种磷酸异构体形式存在，并且可能在CDK5下游受到磷酸化介导的调控。这些发现为进一步研究Numb磷酸化在调节其亚细胞定位、蛋白质相互作用和功能方面的复杂作用提供了基础。所有质谱数据已存入ProteomeXchange，标识符为PXD000997（http://proteomecentral.proteomexchange.org/dataset/PXD000997）。
