TARGETED PROTEOMIC STUDY OF THE CYCLIN-CDK MODULE

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。 酿酒酵母芽生细胞的细胞分裂周期主要由一个细胞周期蛋白依赖性蛋白激酶(CDK)驱动，当它与九个细胞周期蛋白亚基中的一个结合时，它就会变得活跃。这些细胞周期蛋白-CDK复合体的大多数特定结合物和底物仍然难以捉摸，尽管它们的阐明对于全面了解细胞周期是必不可少的。在这里，我们报告了一项有针对性的蛋白质组学研究的结果，该研究确定了许多与特定周期蛋白-CDK复合体相关的蛋白质。这些包括磷酸化底物、泛素化-降解途径中涉及的蛋白质、接头蛋白和抑制物。我们调查并确认了其中一些相互作用的特异性和生物学相关性。我们证明，这种研究生物模块内蛋白质关联的方法允许检测到许多新的关联。我们的数据包括了许多在以前的蛋白质组研究中遗漏的关联，并表明即使是瞬时和动态的相互作用也可以通过基于质谱学的靶向蛋白质组方法来检测。这项工作发表在ArChambault V，Chang EJ，Drapkin BJ，Cross FR，Chait BT，Rout MP，对Cyclin-CDK模块Mol Cell的靶向蛋白质组学研究。2004年，14699-711。我们目前正在过去几年高度优化的条件下重新审视这项实验，并使用时间分辨蛋白质组学和优化的质谱学读数。在这些新的条件下，我们希望分离和鉴定大量新的CDK底物。

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. The cell division cycle of the budding yeast Saccharomyces cerevisiae is mainly driven by one Cdk (Cyclin-Dependent Kinase), which becomes active when bound to one of nine cyclin subunits. The majority of the specific associates and substrates for these cyclin-Cdk complexes remain elusive, though their elucidation is essential for a full understanding of the cell cycle. Here we report the results of a targeted proteomics study that identified numerous proteins associated with particular cyclin-Cdk complexes. These included phosphorylation substrates, proteins involved in the ubiquitination-degradation pathway, adaptor proteins and inhibitors. We investigated and confirmed the specificity and biological relevance of some of these interactions. We demonstrate that this approach for studying protein associations within a biological module allowed the detection of many new associations. Our data includes many associations that were missed in previous proteome-wide studies, and shows that even transient and dynamic interactions can be detected by mass spectrometry-based targeted proteomic approaches. This work was presented in Archambault V, Chang EJ, Drapkin BJ, Cross FR, Chait BT, Rout MP, Targeted proteomic study of the cyclin-Cdk module Mol Cell. 2004, 14, 699-711. We are currently revisiting this experiment under conditions that have been highly optimized over the past several years, as well as using time-resolved proteomics and optimized mass spectrometric readout. Under these new conditions, we hope to isolate & identify a large number of new substrates of Cdk.