The objective of this proposal is to identify and characterize the effector pathways required for Rac1-mediated biological activities. Rac1 is a member of the Ras superfamily of small guanine nucleotide binding proteins. Rac1 is involved in cytoskeletal rearrangements in fibroblasts and, in addition, has been implicated in cellular processes such as transcriptional control, mitogenesis, transformation, and invasiveness. Little is known about the signal transduction pathways mediating these events and, in particular, the effectors by which Rac1 mediates these activities remain poorly characterized. A detailed biochemical and genetic characterization of the known Rac1 effectors is required. The applicant has identified two novel targets of Rac1, designated POR1 and POR2, and has demonstrated that POR1 plays a role in cytoskeletal rearrangement. The project examines the role of POR1 and 2 in the signal transduction pathways downstream from Rac1.
The specific aims are: 1) To investigate the function of POR1 in cytoskeletal rearrangements elicited by Rac1. 2) To assess whether POR1 involvement in morphological changes contributes to the general phenomenon of transformation or metastasis. 3) To examine the role of POR2 in Rac1-induced biological activities. The project presents genetic, biochemical and molecular/cell biological approaches to study the functional role of POR1 and POR2 in Rac1 elicited responses. The potential interaction of POR1 with actin and actin binding proteins will be examined biochemically and by immunofluorescence microscopy. The yeast two-hybrid technique as well as co-immunoprecipitation will be used to look for proteins interacting with POR1. The involvement of POR1 in processes such as transformation, mitogenesis, invasiveness, and mestastasis will be investigated. For the functional characterization of POR2, assays similar to those used for POR1 will be used. A panel of Rac1 mutants with selective binding profiles towards the Rac1 targets will be used to define the role of POR1 and POR2 and other Rac1 targets in the cellular responses triggered by Rac1. The health-relatedness of this project is that by defining the functions of POR1 and 2, new insights may be obtained regarding the molecular mechanisms by which Rac1 mediates its cellular responses, including cellular transformation and metastasis.
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