Mechanism for repression of cyclin A-CDK activity in early G1 phase of mammalian cell cycle

哺乳动物细胞周期早期 G1 期细胞周期蛋白 A-CDK 活性的抑制机制

• 批准号: 11660328
• 负责人: CHIBAZAKURA taku
• 金额: $ 2.11万
• 依托单位: Tokyo University of Agriculture
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660328/
• 关键词: mammalian cell culture; cell cycle; cyclin A; cyclin-dependent kinase (CDK); M; G1 transition; protein degradation; CDK inhibitor; P107; サイクリン依存性キナーゼ(CDK)

Destruction of cyclin A during mitotic (M) phase depends on a destruction box (D box) which is conserved among A- and B-type cyclins. We have constructed a D-box mutant of human cyclin A and investigated its fate and function during the cell cycle in mouse fibroblasts. When expressed at a nearly physiological level, this D-box mutant was stabilized during the mitosis and subsequent entry into G1 phase but did not interfere the progression of M-to-G1 transition. Thus the destruction of cyclin A is not essential for the M-to-G1 transition. Even though the D-box mutant of cyclin A was stable, we found its associated cyclin-dependent kinase (CDK) activity was downregulated at the conclusion of mitosis. The stabilized cyclin A-associated CDK activity was also repressed during the M-to-G1 transition in fibroblasts derived from mice nullizygous for CDK inhibitors p21 and/or p27, indicating that neither p21 nor p27 is essential for the repression. In addition, phosphoamino acid analyses strongly suggested that CDKs associated with the D-box mutant cyclin A are not inactivated by phosphorylation at tyrosines during the M-to-G1 transition. We found that an Rb-family tumor suppressor p107 binds to the D-box mutant cyclin A at the early G1 phase when both p21 and p27 are missing, and that the stabilized cyclin A-associated CDK activity is deprepressed in p21-/-p27-/-p107-/- fibroblasts. These results suggest that p21 and p27 function as primary inhibitors and p107 serves as a secondary inhibitor in the downregulation of the stabilized cyclin A-associated CDK during the M-to-G1 transition. This alternative downregulation pathway might be a "fail-safe" mechanism which maintains normal progression through the cell cycle independent of the cyclin destruction pathway.

有丝分裂(M)期细胞周期蛋白A的破坏依赖于A-型和B-型细胞周期蛋白之间保守的破坏盒(D盒)。我们构建了人细胞周期蛋白A的D盒突变体，并研究了其在小鼠成纤维细胞细胞周期中的命运和功能。当表达在接近生理水平时，该D-box突变体在有丝分裂和随后进入G1期时稳定下来，但不干扰M-G1转变的进程。因此，细胞周期蛋白A的破坏对于M向G1的转变不是必需的。尽管Cyclin A的D-box突变体是稳定的，但我们发现在有丝分裂结束时，其相关的细胞周期蛋白依赖性激酶(CDK)活性下调。在CDK抑制剂p21和/或p27无效的小鼠成纤维细胞中，稳定的周期蛋白A相关的CDK活性在M-G1转变过程中也被抑制，表明p21和p27都不是抑制所必需的。此外，磷酸氨基酸分析强烈表明，与D-box突变体Cyclin A相关的CDK在M-G1转变过程中不会因酪氨酸的磷酸化而失活。我们发现，当p21和p27都缺失时，RB家族肿瘤抑制因子p107在早期G1期与D盒突变体细胞周期蛋白A结合，并且稳定的细胞周期蛋白A相关CDK活性在p21-/-p27-/-p107-/-成纤维细胞中被抑制。这些结果表明，p21和p27在M-G1期稳定的细胞周期蛋白A相关的CDK的下调中起主要抑制作用，而p107起辅助抑制作用。这种可供选择的下调途径可能是一种“故障安全”机制，在细胞周期中维持正常的进程，而不依赖于周期蛋白破坏途径。

项目成果

Chibazakura, T. and Yoshikawa, H.: "A charged amino acid cluster in the C-terminal domain of human cyclin A is required for activation of cyclin-dependent kinase"J. Agric. Sci. TUA. 46. 28-32 (2001)

Chibazakura, T. 和 Yoshikawa, H.：“人细胞周期蛋白 A C 末端结构域中的带电氨基酸簇是细胞周期蛋白依赖性激酶激活所必需的”J.

Chibazakura, T., Yoshikawa, H.: "A charged amino acid cluster in the C-terminal domain of human cyclin A is required for activation of cyclin-dependent kinase"J. Agric. Sci. TUA. 46. 28-32 (2001)

Chibazakura, T., Yoshikawa, H.：“人细胞周期蛋白 A C 末端结构域中的带电氨基酸簇是细胞周期蛋白依赖性激酶激活所必需的”J.

Chibazakura,T.and Yoshikawa,H.: "A charged amino acid cluster in the C-terminal domain of human cyclin A is required for activation of cyclin-dependent kinase."J.Agric.Sci.TUA (in press). (2001)

Chibazakura,T. 和 Yoshikawa,H.：“人细胞周期蛋白 A C 末端结构域中的带电氨基酸簇是细胞周期蛋白依赖性激酶激活所必需的。”J.Agric.Sci.TUA（出版中）。