Abstract:
:Small GTPases of the Ras and Rho families are among the most studied signaling proteins and represent promising therapeutic targets for human neoplastic disease. Despite the high level of interest in these proteins, direct analysis of most aspects of Ras protein biology in living cells has not been possible, because much of the details of Ras signaling cannot be studied in vitro but requires simple cell-based assays. Here we describe a strategy for directly analyzing Ras signaling pathways in living cells using protein-fragment complementation assays (PCA) based on fragments of intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from Ras proteins, as well as complexes of Ras proteins with regulator and effector proteins. We describe high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity (high-content assays). Spatial and temporal changes in response to perturbations (chemical, siRNA, hormones) allow for delineation of Ras signaling networks and a general and high-throughput approach to identify drugs that act directly or indirectly on Ras pathways.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Westwick JK,Michnick SWdoi
10.1016/S0076-6879(05)07032-1subject
Has Abstractpub_date
2006-01-01 00:00:00pages
388-401eissn
0076-6879issn
1557-7988pii
S0076-6879(05)07032-1journal_volume
407pub_type
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