CHARACTERIZATION OF NUCLEATION PROMOTING FACTOR PROTEIN COMPLEXES BY MASS SPEC

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。 肌动蛋白细胞骨架在细胞运动、膜运输和胞质分裂等基本细胞过程中起着至关重要的作用。这些过程对正常的胚胎发育、免疫系统功能和组织修复非常重要，也会导致炎症、心血管疾病、癌症转移和微生物感染等疾病。为了实现肌动蛋白的功能，细胞精确地控制肌动蛋白聚合的成核和由此产生的细丝的组织。我们工作的目标是了解细胞中关键的肌动蛋白成核和组织因子之一Arp2/3复合体的功能和调节。这种蛋白质复合体将肌动蛋白细丝聚合成Y-分支网络，参与片状脂虫的突起、吞噬、内吞和膜运输。Arp2/3复合体的激活需要一种名为核化促进因子(NPF)的蛋白质，哺乳动物细胞中有几种NPF，每一种都能指导Arp2/3复合体参与特定的细胞过程。NPF本身受其他细胞骨架蛋白和信号转导分子的调节，这些分子协调它们在空间和时间上的活动。尽管NPF在调节细胞内肌动蛋白成核方面具有重要作用，但人们对NPF是否存在于一个或多个蛋白质复合体中，哪些其他蛋白质组装成这些复合体，以及这些复合体的活性如何受到磷酸化的调控知之甚少。本项目的目标是利用质谱仪鉴定从哺乳动物细胞中提纯的含NPF的蛋白质复合体的蛋白质组分，并绘制这些复合体各组分中潜在的调节磷酸化位点。从长远来看，这些实验将有助于阐明在细胞运动、膜运输和细胞分裂等过程中，肌动蛋白的核和组织是如何受到控制的。了解这些机制可能最终导致诊断和治疗炎症性疾病、心血管疾病、癌症转移以及细菌和病毒感染的新方法。

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 actin cytoskeleton plays an essential role in basic cellular processes including locomotion, membrane transport, and cytokinesis. These processes are important for normal embryonic development, immune system function and tissue repair, and also contribute to illnesses such inflammatory disease, cardiovascular disease, cancer metastasis, and microbial infection. To enable actin function, cells precisely control the nucleation of actin polymerization and the organization of the resulting filaments. The goal of our work is to understand the function and regulation of one of the cell's key actin nucleating and organizing factors, the Arp2/3 complex. This protein complex polymerizes actin filaments into Y-branched networks that participate in lamellipodia protrusion, phagocytosis, endocytosis and membrane transport. Activation of the Arp2/3 complex requires proteins called nucleation promoting factors (NPFs), of which there are several in mammalian cells, each acting to direct the Arp2/3 complex to participate in a particular cellular process. NPFs are themselves regulated by other cytoskeletal proteins and signal transduction molecules that coordinate their activities in space and time. Despite the key importance of NPFs in regulating actin nucleation in cells, little is known about whether each exists in one or more protein complexes, what other proteins assemble into these complexes, and how the activity of these complexes is regulated by phosphorylation. The goal of this project is to use mass spectrometry to identify the protein components of NPF-containing protein complexes purified from mammalian cells, and to map potential regulatory phosphorylation sites in the components of these complexes. In the long term, these experiments will help elucidate how actin nucleation and organization is controlled in cells during processes such as cell locomotion, membrane transport, and cell division. Understanding these mechanisms may ultimately lead to new approaches to diagnose and treat inflammatory disease, cardiovascular disease, cancer metastasis, and bacterial and viral infection.