SH2/SH3 adaptors of the Crk and Nck families transduce signals by bringing together tyrosine-phosphorylated proteins that bind their SH2 domains with downstream effectors that bind their SH3 domains. These adaptors play a critical role in regulating a variety of normal cell behaviors such as adhesion and motility, while abnormal signaling by Crk can induce oncogenic transformation and may play a role in human cancer. Despite their central role in signaling, understanding their mechanism of action is challenging because they bind to many proteins in the cell with similar affinity, and their activities are highly dependent on their subcellular localization and the local concentration of potential binding partners. We propose an interdisciplinary research plan, using approaches ranging from quantitative computational modeling, phosphoproteomics, live-cell imaging, and in vivo tumorigenicity assays, to reveal in molecular detail the mechanism of action of Crk and Nck adaptors in important biological activities. Specifically, the experiments proposed here will 1.) elucidate the molecular basis for the positive role for the Sos1 guanine nucleotide exchange factor (GEF) and the negative role for Abl tyrosine kinases in transformation by Crk;2.) elucidate the mechanisms whereby Abl-Crk and Abl-Nck complexes regulate local actin cytoskeletal organization, such as the balance between filopodia and lamellipodia in spreading cells;and 3.) use a spatial computational model of actin dynamics, together with experimental methods to manipulate local concentrations of Nck and other signaling proteins in living cells, to obtain a quantitative understanding o how Nck controls the local state of actin polymerization in the cell.
Signals mediated by tyrosine phosphorylation control a host of important biological activities, and when dysregulated can cause human diseases such as cancer and immune disorders. SH2/SH3 adaptors play a central role in tyrosine kinase signaling, but their specific mechanism of action is poorly understood. The proposed studies will provide specific, mechanistic details about adaptor function that will provide insight into basic biological processes as well as how these processes can be subverted in disease.
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