CHARACTERIZATION OF NUCLEATION PROMOTING FACTOR PROTEIN COMPLEXES BY MASS SPEC

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。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 研究中心，而研究中心不一定是研究者所在的机构。 肌动蛋白细胞骨架在基本的细胞过程中起着重要的作用，包括运动，膜运输和胞质分裂。 这些过程对于正常的胚胎发育、免疫系统功能和组织修复都很重要，也会导致炎症性疾病、心血管疾病、癌症转移和微生物感染等疾病。为了使肌动蛋白发挥功能，细胞精确地控制肌动蛋白聚合的成核和所产生的细丝的组织。 我们工作的目标是了解细胞的关键肌动蛋白成核和组织因子之一Arp 2/3复合物的功能和调节。 这种蛋白质复合物将肌动蛋白丝聚合成Y分支网络，参与板状伪足突出、吞噬作用、内吞作用和膜运输。 Arp 2/3复合物的激活需要称为成核促进因子（NPF）的蛋白质，其中在哺乳动物细胞中有几种，每种蛋白质都能指导Arp 2/3复合物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，这些分子在空间和时间上协调它们的活动。 尽管NPF在调节细胞中肌动蛋白成核方面至关重要，但人们对每种NPF是否存在于一种或多种蛋白质复合物中、还有哪些其他蛋白质组装成这些复合物以及这些复合物的活性如何通过磷酸化来调节知之甚少。本项目的目标是利用质谱鉴定从哺乳动物细胞中纯化的含NPF的蛋白质复合物的蛋白质组分，并绘制这些复合物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肌动蛋白成核和组织是如何在细胞运动，膜运输和细胞分裂过程中控制的。 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.