| Budget Amount *help |
¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
The degradation of mitotic cyclins plays an essential role in cell-cycle progression during mitosis. Mitotic cyclins A and B are degraded by ubiquitin-dependent proteolysis and its process requires the N-terminal domain of mitotic cyclins. Using the N-terminus of cyclin A1 in a 2-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to S.cerevisiae Dsk2. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A.XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A.The addition of bacterially-expressed XDRP1 protein to frog egg extract inhibited the Ca2+-induced degradation of cyclin A, but not that of cyclin B.The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division. These results suggest that XDRP1 is a negative regulator of degradation of cyclin A.
XDRP1 contains a ubiquitin-like domain in its N-terminus and shows significant homology to S.cerevisiae Dsk2. Based on above evidence, we investigate the function of XDRP1/Dsk2 on ubiquitin-dependent proteolysis in yeast system. When overexpressed in yeast cells, Dks2 caused to accumulate polyubiquitination. Supprcssors of DSK2 toxicity in yeast were identified as mutations in proteasome subunits ; PRE2 in core subunit, and RPN1 in regulatory subunit, indicating genetic interaction between Dsk2 and proteasome. The results suggest that Dsk2 have a regulatory role in ubiquitin-proteasome degradation pathway.
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