|Budget Amount *help
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2006: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2004: ¥5,600,000 (Direct Cost: ¥5,600,000)
Structure-function studies of protein domains are always very productive in a sense that one study on a representative protein domain makes it possible to infer the structures and functions of many family proteins that contain the protein domain. The structure determination of SH2 and SH3 domains in the early 1990s represents the first good example. We determined the first three-dimensional structure of the PX domain. The present study aimed at the systematic detection of the interaction of the PX domains with the SH3 domains in yeast,. Since many PX and SH3 domains are encoded in eukaryotic genomes, the establishment of the PX-SH3 interactions would have impacts on the field of cell biology.
The PX-SH3 interaction was probable three years ago when this project started, but. we must, conclude that. the PX-SH3 interaction is unlikely to exist, considering the negative results from the pull-down assay and yeast two-hybrid assay using several yeast PX and SH3 domains in the present, study.
We also carried out NMR titration analysis on the most likely combination, SNX3-PX and Rvs167-SH3,suggested by a different, group's systematic yeast two-hybrid assay. No interaction was detected despite of the sensitivity of the NMR method toward the weak interactions.
At the third year of the project, we selected a new domain, PXA domain (PX associated domain), as a target of the PX domain. The PXA domain is defined simply on the sequence homology, and no structural and functional information is available. We succeeded to prepare the PXA domains from human SNX13 and SNX14, and detect, the interaction between the PXA and PX domains from SNX14. Now we postulate that a protein is in a closed conformation in a resting state due to the PX-PXA interaction. The protein becomes opened in an activated state, and the exposed PX domain recruits the protein onto the membrane using its affinity for phosphatidylinositolphosphates (PIPs). This mechanism enables the another domain between the PX and PXA domains in the primary sequence, for example, RGS domain, to be located near the membrane and fulfill the function. Less