Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Montpellier Cedex, France.
Oncogene. 2008 Sep 18;27(42):5554-66. doi: 10.1038/onc.2008.167. Epub 2008 May 26.
Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.
Chfr 是一种检查点蛋白，在细胞周期进程和肿瘤抑制中发挥重要作用，尽管其确切作用和调节机制尚不清楚。先前的研究利用 Chfr 的过表达来确定该蛋白在体内的信号通路。在这项研究中，我们通过使用三种针对 Chfr 的不同抗体，证明内源性和高度过表达的异位 Chfr 蛋白在细胞中的定位和调节方式不同。内源性和低表达的异位 Chfr 位于细胞质中，并在有丝分裂期间定位于纺锤体。异位 Chfr 的高表达与该蛋白定位到核/PML 体的转移以及细胞增殖的阻断相关。此外，内源性和低表达的异位 Chfr 在整个细胞周期中是稳定的，而当高度表达时，异位 Chfr 以自主泛素化和蛋白酶体依赖的方式在 S-G2/M 期被积极降解。双杂交筛选鉴定出 TCTP 可能是 Chfr 的相互作用伙伴。用内源性蛋白进行的生化分析证实了这种相互作用，并鉴定出β-微管蛋白是 Chfr 的另一个伴侣，支持 Chfr 在有丝分裂纺锤体中的定位。Chfr-TCTP 相互作用在整个细胞周期中是稳定的，但它可以被微管的完全解聚所减弱，这为 Chfr 可能是检测微管破坏并随后激活前期检查点的传感器提供了一种可能的机制。
