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.