Cheng M, Sexl V, Sherr C J, Roussel M F
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.
Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1091-6. doi: 10.1073/pnas.95.3.1091.
A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.
在锌诱导型启动子的控制下，在NIH 3T3成纤维细胞中合成了有组成活性的丝裂原活化蛋白激酶激酶（MEK1）。用锌处理血清饥饿细胞可激活细胞外信号调节蛋白激酶（ERK）并诱导细胞周期蛋白D1的表达。新合成的细胞周期蛋白D1与细胞周期蛋白依赖性激酶4（CDK4）组装形成全酶复合物，该复合物对视网膜母细胞瘤蛋白的磷酸化效率低下。MEK1/ERK途径的激活既未触发细胞周期蛋白依赖性激酶抑制剂激酶抑制蛋白1（p27（Kip1））的降解，也未导致细胞周期蛋白E和A依赖性CDK2的激活，并且此类细胞未进入细胞分裂周期的DNA合成（S）期。相比之下，在经过基因工程改造以异位过表达细胞周期蛋白D1和CDK4亚基的细胞中，锌诱导的活性MEK1产生的细胞周期蛋白D依赖性视网膜母细胞瘤蛋白激酶活性水平接近血清刺激细胞中达到的水平。在这种情况下，p27（Kip1）被募集到含有细胞周期蛋白D1的复合物中；细胞周期蛋白E和A依赖性CDK2复合物被激活；血清饥饿细胞进入S期。因此，尽管p27（Kip1）的活性通常通过血清依赖性降解过程被消除，但过表达的细胞周期蛋白D1-CDK复合物通过化学计量机制隔离了p27（Kip1）并降低了有效抑制阈值。这些细胞中的一部分完成了S期并进行了分裂，但它们无法持续增殖，这表明其他血清反应性因子最终成为细胞周期进程的限速因素。因此，MEK/ERK途径不仅在转录水平上起作用以诱导细胞周期蛋白D1基因，而且在翻译后发挥功能，调节细胞周期蛋白D1与CDK4的组装，从而有助于消除p27（Kip1）介导的抑制作用。
