Construction of a Mitogenesis-Coupled Apoptosis Molecular Device
Abstract
In this grant proposal, we proposed to construct Ras/Raf-based MCAMD, i.e. fuse one subunit of caspase-3 to Raf and the other subunit to Ras. Once Ras interacts with Raf in response to mitogenic signal, the two subunits of caspase-3 that are fused to Ras and Raf will be brought together to form mature active caspase-3, thus cellular apoptosis will be initiated. During eighteen months, we constructed plasmids that are required for testing MCAMD. We characterized the interaction of separated C3SS and C3LS and established a base for application of Caspase-3 in construction of MCAMD. We tested initial cellular reactions of MCAMD and found that C3SS-Ras could not co-express with Raf-RBD-C3LS, suggesting that C3SS-Ras and Raf-RBD-C3LS might form active Caspase-3 and cause a rapid cell death. However, because this research is novel and we did not have experience in construction and testing of MCAMD, we met some unexpected problems, such as weak interaction of wild type Ras with Raf-RBD that causes constitutive activation of MCAMD and makes that MCAMD cannot sense mitogenic signaling. Future studies will include: (a) to overcome signaling switch problem of the MCAMD; (b) to assay caspase-3 activity and apoptosis upon co-transfection of tetracycline-inducible expression of C3SS-RasWT and Raf-RBD-C3LS; and (c) to test MCAMD in prostate cancer cells.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2006
- Accession Number
- ADA462387
Entities
People
- Wannian Yang