爪蟾 Cip 型 CDK 抑制剂 p16Xic2 的蛋白水解由 PCNA 结合和 CDK2 磷酸化调节。

Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation.

机构信息

Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

出版信息

Cell Div. 2013 Apr 22;8(1):5. doi: 10.1186/1747-1028-8-5.

DOI:10.1186/1747-1028-8-5

PMID:23607668

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3655096/

Abstract

BACKGROUND

Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3.

METHODS

We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.

RESULTS

Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.

CONCLUSIONS

During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.

摘要

背景

细胞分裂受细胞周期蛋白依赖性激酶（CDK）与细胞周期蛋白的正调控，受 CDK 抑制剂的负调控。在青蛙 Xenopus laevis 中，已经描述了三种类型的 CDK 抑制剂：p27Xic1（Xic1），它与哺乳动物的 p21Cip1 和 p27Kip1 具有序列同源性，p16Xic2（Xic2），它与 p21Cip1 具有序列同源性，以及 p17Xic3（Xic3），它与 p27Kip1 具有序列同源性。虽然过去的研究表明，在 DNA 聚合酶切换过程中，Xic1 依赖于 DNA、增殖细胞核抗原（PCNA）和泛素连接酶 CRL4Cdt2 进行蛋白质周转，但是对于调节 Xic2 或 Xic3 的过程知之甚少。

方法

我们使用 Xenopus 间期卵提取物作为模型系统，研究 Xic2 的蛋白水解和磷酸化调节。

结果

我们的研究表明，在 DNA 复制起始期间合成引物后，Xic2 被靶向进行 DNA 和 PCNA 依赖性泛素介导的蛋白水解，并且 Cdt2 可以促进 Xic2 周转。此外，在间期，Xic2 被 CDK2 在 Ser-98 和 Ser-131 处非依赖性磷酸化，抑制 Xic2 周转。在双链 DNA 末端存在的情况下，Xic2 也被咖啡因敏感激酶在 Ser-78 和 Ser-81 处磷酸化，但这种磷酸化不会改变 Xic2 周转。相反，在存在或不存在 DNA 的情况下，Xic3 在 Xenopus 间期卵提取物中稳定，并且没有表现出指示磷酸化的移位。

结论

在间期，Xic2 被靶向进行 DNA 和 PCNA 依赖性蛋白水解，该蛋白水解受 CDK2 磷酸化的负调控。在对 DNA 损伤的反应中，Xic2 可能通过咖啡因敏感激酶的磷酸化被替代调节。我们的研究表明，三种类型的 Xenopus CDK 抑制剂，Xic1、Xic2 和 Xic3 似乎受到独特的调节，这可能反映了它们在细胞分裂或青蛙早期发育中的特殊作用。