Huang Sui, Ingber Donald E
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Exp Cell Res. 2002 May 1;275(2):255-64. doi: 10.1006/excr.2002.5504.
Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G(1) and entry into S phase of the cell cycle. Pharmacological disruption of CSK integrity inhibits early mitogenic signaling to the extracellular signal-regulated kinase (Erk) subfamily of the mitogen-activated protein kinases (MAPKs) and arrests the cell cycle in G(1). Here we show that this block of G(1) progression is not simply a consequence of inhibition of the MAPK/Erk pathway but instead it reveals the existence of a discrete CSK-sensitive checkpoint. Use of PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at progressive time points in G(1) revealed that the requirement for MAPK/Erk activation lasts only to mid-G(1), while the actin CSK must remain intact up to late G(1) restriction point, R, in order for capillary endothelial cells to enter S phase. Additional analysis using Cyto D pulses defined a narrow time window of 3 h just prior to R in which CSK integrity was shown to be critical for the G(1)/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27(Kip1), independent of cell cycle phase, suggesting that these changes resulted directly from CSK disruption rather than from a general cell cycle block. Together, these data indicate the existence of a distinct time window in late G(1) in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G(1)/S boundary and, hence, promote cell growth.
细胞在细胞外基质上的铺展以及肌动蛋白细胞骨架（CSK）的相关变化是细胞通过G1期并进入细胞周期S期所必需的。CSK完整性的药理学破坏会抑制向丝裂原活化蛋白激酶（MAPK）的细胞外信号调节激酶（Erk）亚家族的早期促有丝分裂信号传导，并使细胞周期停滞在G1期。在这里，我们表明G1期进程的这种阻滞不仅仅是MAPK/Erk途径抑制的结果，相反，它揭示了一个离散的CSK敏感检查点的存在。在G1期的不同时间点使用PD98059抑制MAPK/Erk和细胞松弛素D（Cyto D）破坏肌动蛋白CSK，结果表明对MAPK/Erk激活的需求仅持续到G1期中期，而肌动蛋白CSK必须在G1期晚期限制点R之前保持完整，以便毛细血管内皮细胞进入S期。使用Cyto D脉冲进行的进一步分析定义了紧接R之前的一个3小时的狭窄时间窗口，其中CSK完整性对于G1/S转变至关重要。Cyto D处理导致细胞周期蛋白D1蛋白下调和细胞周期蛋白依赖性激酶抑制剂p27（Kip1）积累，与细胞周期阶段无关，这表明这些变化直接源于CSK破坏而非一般的细胞周期阻滞。总之，这些数据表明在G1期晚期存在一个独特的时间窗口，其中CSK引发的信号独立于早期MAPK/Erk信号起作用，以驱动细胞周期机制通过G1/S边界，从而促进细胞生长。
