| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Dominant active mutations of the ras gene induce malignant cellular transformation, while overexpression of the rap1A gene induces reversion of the ras-transformed cells. These genes encode similar small GTP-binding proteins, Ras and Rap1A.We had identified two novel Ras/Rap1A-binding proteins, a phospholipase C (Ce-PLC-ε) and a GDP/GTP exchange factor (Ce-RA-GEF), from nematode C.elegans. Both of these molecules possessed the Ras/Rap1A-associating (RA) domain (s). We had also isolated partial cDNAs for their human homologs (Hs-PLC-ε, Hs-RA-GEF). In the present study, we obtained full-length cDNAs and examined their functions.
Hs-PLC-ε associated with Ras in a GTP-dependent manner in vitro. Co-expression of an activated Ras mutant with Hs-PLC-ε induced its translocation from the cytosol to the plasma membrane. Upon stimulation with EGF, similar translocation was observed, which is inhibited by co-expression of dominant negative Ras. In a liposomebased reconstitution assay, the phosphati
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dylinositol 4,5-bisphosphate-hydrolyzing activity of Hs-PLC-ε was stimulated in vitro by Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of Hs-PLC-ε.
Ce-RA-GEF and Hs-RA-GEF possessed a PDZ domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cAMP/cGMP-binding domain, which turned out to be incapable of binding cAMP or cGMP.Although the REM domain and GEF domains are conserved with other GEFs acting on Ras family proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A.These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution. Less
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