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標題: 73位置絲胺酸的磷酸化降低酵母菌Rad23與26S蛋白酶體的結合能力及其所調控的蛋白質降解
Phosphorylation of Serine-73 decreases the association of yeast Rad23 with the 26S proteasome and Rad23-mediated protein degradation
作者: 梁瑞岳
Liang, Ruei-Yue
關鍵字: Rad23
protein degradation
出版社: 生物醫學研究所
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摘要: Rad23是一個具有高度保留性的蛋白,並且對於泛素系統所調控的蛋白質降解和細胞內的核甘酸修補機制是重要的。然而,細胞是透過何種重要訊號來調控 Rad23的功能目前還不清楚,為了研究細胞內訊號是如何調控Rad23,利用定位點突變技術及體外激酶實驗證實Rad23的73號位置絲胺酸是可以被激酶Rad53所磷酸化。此外,不管在活體內或活體外的實驗,73位置模擬磷酸化的突變株S73D降低了Rad23和26S蛋白水解酶複合體的結合能力。有趣的是,當細胞處理staurosporine來抑制磷酸化的現象,會增加Rad23和26S蛋白水解酶複合體的結合能力。另外經由泛素系統所降解的蛋白質Ub-Proline-β-galactosidase (Ub-Pro-β-gal),在表現S73D的突變株中,其穩定度是明顯的增加。而在Rad23功能性的分析上發現S73A/D的突變株並不影響細胞對UV的敏感度且對於Rad4的穩定度也不受影響。基於以上的研究結果,我們推測Rad23的73號位置絲胺酸磷酸化會降低Rad23和26S蛋白水解酶複合體的結合能力,進而影響到其所調控的蛋白質降解,但其調控的分子機制則須更進一步的研究探討。
Rad23 is an evolutionarily conserved protein that is important for ubiquitin-proteasome system (UPS) and nucleotide excision repair (NER). However, the significance signaling regulation of Rad23 function is still unclear. Site-directed mutagenesis and in vitro kinase assay proved that Serine-73 is phosphorylated by Rad53. In addition, phosphorylation-mimic S73D mutant decreased the association of Rad23 to the 26S proteasome in vivo and in vitro. Interestingly, staurosporine increased the Rad23 and 26S proteasome interaction. Ub-Proline-β-galactosidase (Ub-Pro-β-gal), a model substrate of the ubiquitin/ proteasome pathway, was significantly stabilized in S73D expressing strain. Furthermore, functional analysis showed that S73A/D mutants were similar to wild-type in UV sensitivity and Rad4 stability. Based on these data, we suggest that phosphorylation of Serine-73 decreases the association of Rad23 with the 26S proteasome and affects the Rad23-mediated proteolysis. However, the molecular mechanism for S73 phosphorylation by signaling will be further investigated.
其他識別: U0005-2907201016353000
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