Fukuchi Kunihiko, Hagiwara Tamio, Nakamura Kentarou, Ichimura Sachiko, Tatsumi Kouichi, Gomi Kunihide
Department of Biochemistry, Showa University, School of Medicine, Tokyo 142-8555, Japan.
Biochem Biophys Res Commun. 2002 Apr 26;293(1):120-5. doi: 10.1016/S0006-291X(02)00198-5.
The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.
细胞周期蛋白激酶抑制剂p21的表达受泛素 - 蛋白酶体蛋白降解系统以及转录调控。一般来说，泛素化受底物磷酸化的调节。在本研究中，我们确定了p21中负责泛素化调节的区域。由于p21的磷酸化位点分布在C末端区域，我们构建了连续的C末端截短片段，并在真核细胞中检测了它们的泛素化情况。在1 - 164（全长）和1 - 157片段中观察到了相同效率的泛素化，但在1 - 147片段中未观察到。1 - 147片段中缺乏泛素化不太可能是由于154位赖氨酸残基的去除，因为p21 K154R突变体与全长p21一样有效地被泛素化。此外，p21的148 - 157缺失形式与1 - 147片段一样未被泛素化。因此，C末端148 - 157区域本身虽不是泛素化位点，但应包含p21有效泛素化的关键调节区域。
