Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression
Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression
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DOI：
10.1074/jbc.m310383200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Baldassare, JJ
中科院分区：
文献类型：
--
作者：
Keenan, SM;Lents, NH;Baldassare, JJ
研究表明，CDK2-Cyclin E在视网膜母细胞瘤蛋白失活和细胞周期G(1)-S期进展中起关键作用。磷脂酰肌醇3-羟基激酶抑制剂LY294002已被证明可以抑制α-凝血酶刺激的IIC9细胞(中国仓鼠胚胎成纤维细胞)中细胞周期蛋白D1的积聚和CDK4的活性，从而阻止G(1)的进展。我们先前的结果表明，细胞周期蛋白E的表达挽救了LY294002处理的α-凝血酶刺激的IIC9细胞的S期进展，认为细胞周期蛋白E使CDK4活性对于G(1)进展是必不可少的。在这项工作中，我们研究了在缺乏CDK4-细胞周期蛋白D1活性的情况下，α-凝血酶诱导的CDK2-细胞周期蛋白E活性灭活pRB的能力。我们报道，在缺乏CDK4-细胞周期蛋白D1活性的情况下，CDK2-细胞周期蛋白E在体内至少一个残基上磷酸化pRB，并取消pRB与E2F反应元件的结合。我们还发现，在细胞周期蛋白D激酶抑制的、α-凝血酶刺激的细胞中，细胞周期蛋白E的表达挽救了E2F的激活和周期蛋白A的表达。此外，E的表达对E2F活性的挽救、细胞周期蛋白A的表达和DNA的合成都可以被pRb的结构性活性突变体CDK2(D145N)或RB-DeltaCDK的表达所阻断。然而，将已知的4个Cyclin E-CDK2磷酸化位点恢复到Rb-DeltaCDK使其在G(1)晚期失活，如E2F激活、Cyclin A表达和S期进展所检测的那样。这些数据表明，CDK2-Cyclin E在没有CDK4-Cyclin D活性的情况下，可以磷酸化和失活pRb，激活E2F，并诱导DNA合成。
The activation of CDK2-cyclin E in late G(1) phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G(1)-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G(1) progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G(1) progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or Rb-DeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to Rb-DeltaCDK renders it susceptible to inactivation in late G(1), as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.