ALTERED REGULATION OF G(1)-CYCLINS IN SENESCENT HUMAN-DIPLOID FIBROBLASTS - ACCUMULATION OF INACTIVE CYCLIN-E-CDK2 AND CYCLIN-D1-CDK2 COMPLEXES
ALTERED REGULATION OF G(1)-CYCLINS IN SENESCENT HUMAN-DIPLOID FIBROBLASTS - ACCUMULATION OF INACTIVE CYCLIN-E-CDK2 AND CYCLIN-D1-CDK2 COMPLEXES
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DOI：
10.1073/pnas.90.23.11034
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发表时间：
1993-12-01
影响因子：
11.1
通讯作者：
STEIN, GH
中科院分区：
文献类型：
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作者：
DULIC, V;DRULLINGER, LF;STEIN, GH
衰老的人二倍体成纤维细胞不能响应有丝分裂刺激而进入S期。丝裂原刺激的衰老细胞的关键缺陷之一是它们不能磷酸化视网膜母细胞瘤蛋白，该蛋白以其非磷酸化形式充当进入S期的抑制剂。最近的数据表明，受 G1 细胞周期蛋白（D 型和 E 型）调节的细胞周期蛋白依赖性激酶 (Cdks) 负责 G1/S 边界之前视网膜母细胞瘤蛋白的初级磷酸化。令人惊讶的是，我们发现衰老细胞中细胞周期蛋白 E 和细胞周期蛋白 DI 的组成量比静止早期传代细胞高 10 至 15 倍。然而，尽管细胞周期蛋白 E-Cdk2 复合物丰富，但衰老细胞中细胞周期蛋白 E 相关激酶的活性非常低，并且在有丝分裂刺激后并未显着增加。与 G1 期晚期的早期传代细胞相比，衰老细胞主要含有磷酸化不足的细胞周期蛋白 E，以及相应比例更多的未磷酸化和失活的 Cdk2，这可能是激酶活性较低的原因。我们还表明，衰老细胞中的大部分Cdk2与细胞周期蛋白D1复合，但早期传代细胞中则不然。细胞周期蛋白 D1-Cdk2 复合物在衰老细胞中富集数倍，仅包含未磷酸化的 Cdk2。通常在 S 期和 G2 期积累的细胞周期蛋白 A 的量在受刺激的衰老细胞中极低。我们认为，衰老细胞中细胞周期蛋白 E-Cdk2 激酶活性的激活失败可能是这些细胞无法在 G1 期晚期磷酸化视网膜母细胞瘤蛋白的原因，这反过来又可能阻断晚期 G1 基因的表达，例如进入 S 期所需的细胞周期蛋白 A。
Senescent human diploid fibroblasts are unable lo enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent cells is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior to the G1/S boundary. Surprisingly, we found 10- to 15-fold higher constitutive amounts of both cyclin E and cyclin DI in senescent cells compared to quiescent early-passage cells. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase significantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to early-passage cells in late G1 phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin Dl. Cyclin D1-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent cells. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability of these cells to phosphorylate the retinoblastoma protein in late G1 phase, which in turn may block the expression of late G1 genes such as cyclin A that are required for entry into S phase.