Braden Wesley A, Lenihan Jon M, Lan Zhengdao, Luce K Scott, Zagorski William, Bosco Emily, Reed Michael F, Cook Jeanette G, Knudsen Erik S
Department of Cell Biology, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA.
Mol Cell Biol. 2006 Oct;26(20):7667-81. doi: 10.1128/MCB.00045-06. Epub 2006 Aug 14.
The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G1, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.
视网膜母细胞瘤（RB）和p16ink4a肿瘤抑制因子被认为在一条线性通路中发挥作用，而这条通路在大部分人类癌症中功能失活。最近的研究表明，RB在调节S期过程中起着关键作用，以此作为抑制异常增殖和控制基因组稳定性的一种手段。在此，我们证明了p16ink4a在复制控制中具有一种不同于RB的新作用。具体而言，p16ink4a通过在G1期抑制微型染色体维持（MCM）蛋白加载来破坏复制前复合体的组装，而RB则是通过削弱增殖细胞核抗原（PCNA）的功能在S期破坏复制。p16ink4a对复制前复合体的这种影响依赖于RB的存在以及细胞周期蛋白依赖性激酶（CDK）活性的下调。引人注目的是，抑制CDK2活性不足以阻止MCM蛋白加载到染色质上，这支持了一种模型，即多个G1期CDK复合体的复合作用调节复制前复合体的组装。此外，p16ink4a降低了组装因子Cdt1和Cdc6的水平。这两种许可因子的强制表达足以恢复复制前复合体的组装，但由于PCNA功能仍然缺失而未能促进S期进程。综合来看，这些数据表明RB和p16ink4a通过不同的途径发挥作用来抑制复制机制，并提供了证据证明CDK活性的逐步调节在两个离散的执行点与复制机制相互作用。
