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

细胞周期蛋白A在有丝分裂（M）期的破坏依赖于一个在A型和B型细胞周期蛋白中保守的破坏盒（D盒）。我们构建了一个人细胞周期蛋白A的D-box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。当以接近生理水平表达时，这种D盒突变体在有丝分裂和随后进入G1期期间稳定，但不干扰M到G1转变的进程。因此，细胞周期蛋白A的破坏对于M到G1的转变不是必需的。尽管细胞周期蛋白A的D-box突变体是稳定的，但我们发现其相关的细胞周期蛋白依赖性激酶（CDK）活性在有丝分裂结束时下调。稳定的细胞周期蛋白A相关的CDK活性也被抑制在M-到-G1的过渡在成纤维细胞来源于小鼠的CDK抑制剂p21和/或p27无效，表明既不是p21也不是p27是必不可少的镇压。此外，磷酸化氨基酸分析强烈表明，与D盒突变细胞周期蛋白A相关的CDK在M-到-G1过渡期间不被酪氨酸磷酸化灭活。我们发现Rb家族肿瘤抑制因子p107在G1期早期与D-box突变型细胞周期蛋白A结合，此时p21和p27均缺失，并且稳定的细胞周期蛋白A相关的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的功能作为主要的抑制剂和p107作为一个次要的抑制剂，在下调稳定的周期蛋白A相关的CDK在M-到-G1的过渡。这种替代性下调途径可能是一种“故障安全”机制，其通过独立于细胞周期蛋白破坏途径的细胞周期维持正常进展。

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.