| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
NEDD8/Rub1 is a ubiquitin-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all Cullin-family proteins. The latter are essential components of SCF-like ubiquitin ligase complexes, which play critical roles in ubiquitin-mediated proteolysis. Since the role of the NEDD8 conjugation on the SCF complex has been unknown, we investigated the role of the NEDD8 system by genetic and biochemical means.
(1) Analysis of mice deficient in Uba3 gene that encodes a catalytic subunit of NEDDS-activating enzyme. Uba3^<-/-> mice died in utero at peri-implantation stage. NEDDS-activating enzyme. Uba3^<-/-> mice died in utero at peri-implantation stage. Mutant embryos showed selective apoptosis of the inner cell mass, but not of trophoblastic cells. The mutant trophoblastic cells, however, could not enter the S phase of the endoreduplication cycle. This cell-cycle arrest was accompanied with aberrant expressi
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on of Cyclin E and p57^<Kip2>. These results suggested that the NEDD8 system is essential for both mitotic and endoreduplicative cell-cycle progression, β-catenin, a mediator of Wnt/Wingless signaling pathway, which continuously degrades in the cytoplasm through SCF ubiquitin ligase, was also accumulated in the Uba3^<-/-> cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell-cycle progression and morphogenesis, possibly through the function of the Cullin-family proteins.
(2) Genetic analysis of the NEDD8 system in fission yeast. The NEDD8 system is essential for cell viability and function of Pcul (cullin-1 orthologue) in fission yeast. Pcul^<K713R> defective for NEDD8 conjugation lost the ability to complement lethality due to pcul deletion. Forced expression of Pcul^<K713R> or deletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1.
(3) The effect of the NEDD8 system on in vitro ubiquitylation assay of the SCF complex. Using in vitro ubiquitylation assay, we found that NEDD8 modification of the SCF complex enhances the ubiquitylation activity of the SCF complex through the recruitment the ubiquitin-conjugating enzyme Ubc4 (E2). Furthermore, the recruitment of E2 was dependent on the thioester linkage of ubiquitin. Less
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