Okuda A, Ohtsu M, Kimura G
Department of Virology Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Cell Struct Funct. 2001 Aug;26(4):205-14. doi: 10.1247/csf.26.205.
A temperature-sensitive cell-cycle mutant of the 3Y1 rat fibroblast cell line, 3Y1tsD123 has in the D123 gene coding region a point mutation which causes instability of the D123 protein. Temperature-sensitive G1 arrest of the mutant is caused by increased degradation of the D123 protein at restrictive temperature. In this study we found that the selective proteasome inhibitors lactacystin and MG132 inhibited degradation of the mutated D123 protein in cell lines overexpressing the mutated D123 protein, followed by accumulation of a modified form (increased molecular weight other than by ubiquitination) of the D123 protein. Although a temperature-resistant revertant of the mutant had no further mutation in the D123 gene coding region, the modification of the mutated D123 protein was inhibited and the mutated D123 protein was rendered stable. The modification was also inhibited in the hybrid cell lines between the revertant and the cell line overexpressing the mutated D123 protein. These facts imply that the mutated D123 protein receives unidentified modification before degradation in the proteasome, and that the revertant expresses a gene inhibiting this modification.
3Y1大鼠成纤维细胞系的一个温度敏感型细胞周期突变体3Y1tsD123，在D123基因编码区存在一个点突变，该突变导致D123蛋白不稳定。突变体的温度敏感型G1期阻滞是由限制温度下D123蛋白降解增加所致。在本研究中，我们发现选择性蛋白酶体抑制剂乳胞素和MG132可抑制过表达突变型D123蛋白的细胞系中突变型D123蛋白的降解，随后D123蛋白出现一种修饰形式（分子量增加而非泛素化所致）的积累。尽管该突变体的一个温度抗性回复株在D123基因编码区没有进一步的突变，但突变型D123蛋白的修饰受到抑制，突变型D123蛋白变得稳定。在回复株与过表达突变型D123蛋白的细胞系之间的杂交细胞系中，这种修饰也受到抑制。这些事实表明，突变型D123蛋白在蛋白酶体降解之前会受到未明确的修饰，并且回复株表达一种抑制这种修饰的基因。
