| Budget Amount *help |
¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
We analyzed physiological function of POB1, a Ral-downstream protein.
(1) POB1 binds to ASAP1 / PAG and RalBP1 at discrete region.
ASAP1 was identified as a POB1 binding protein by screening mouse brain cDNA library.
A set of deletion mutants of ASAP1 and its human ortholog, PAG2, were synthesized and the mutual binding sites between POB1 and ASAP / PAG2 were determined. POB1 has three praline-rich motifs (amino acid residues 338-345, 374-383, and 422-428). The praline-rich motif 422-428 of POB1 bound to ASAP1/PAG2 on an SH3 domain at Carboxyl terminus of ASAP1 / PAG2 APOB1 mutant, POB1(P423/426A) which had amino acid substitutions of pralines 423 and 426 to alanines, bound to RalBP1,be did not bind to PAG2. This result indicated that PAG2 and RalBP1 bind to POB1 on different regions. Actually PAG2 are RalBP1 on contact with POB1, formed a ternary complex in vivo and in vitro.
(2) POB1 releases the suppression of cell-migration by PAG2.
PAG2 suppressed the accumulation of paxillin to focal adhesion plaques and the cell-migration induced by fibronectin, but did not affect cell adhesion to fibronectin. Wild-type POB1 which bound to PAG2, released the suppression of the paxilli* accumulation and the suppression of the fibronectin-induced cell-migration by PAG2. However, the mutant POB1 (P423 / 426A) which bound to RalBP1 but not to PAG2, did not release the suppression. A PAG2 deletion mutant, PAG2-(1-703), whi* retained Arf GAP activity but lacked the POB1-binding SH3 domain, suppressed the fibronectin-induced cell-migration, are POB1 did not release the suppression.
These results indicate that the interaction between POB1 and PAG2 regulates physiological function of PAG2.
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