Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
PLoS One. 2010 Nov 30;5(11):e15128. doi: 10.1371/journal.pone.0015128.
Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established.
METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes.
CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis.
尽管有丝分裂缺陷蛋白 MAD2B（MAD2L2）被认为通过与 CDC20 和/或 CDH1（FZR1）结合来抑制后期促进复合物（APC），但其在细胞周期控制中的确切作用仍有待确定。
方法/主要发现：我们使用酵母双杂交相互作用陷阱鉴定了人类笼形蛋白轻链 A（CLTA）作为一种新型 MAD2B 结合蛋白。在细胞周期的 G2/M 期，通过 GST 下拉和内源性共免疫沉淀在哺乳动物细胞中建立了直接相互作用。通过随后的共聚焦激光扫描显微镜，我们发现 MAD2B 和 CLTA 在有丝分裂纺锤体上共定位。网格蛋白形成三聚体结构，即网格蛋白三腿体，由三条重链（CLTC）组成，每条重链都有一个相关的轻链。以前的研究表明，这种网格蛋白结构对于通过稳定动粒纤维来发挥有丝分裂纺锤体的功能是必需的。在用 siRNA 介导的 MAD2B 耗竭后，我们发现 CLTA 不再集中在有丝分裂纺锤体上，而是弥散分布在整个细胞中。此外，我们发现染色体错位的百分比明显增加。
结论/意义：以前，我们鉴定 MAD2B 为肾细胞癌（RCC）相关蛋白 PRCC 的相互作用蛋白。此外，我们发现 PRCC 与转录因子 TFE3 的融合在 t(X;1)(p11;q21)-阳性 RCC 中导致这种相互作用的破坏以及随之而来的 MAD2B 不能向核内转移。我们当前的数据表明，MAD2B 在细胞周期的 G2/M 期与 CLTA 相互作用，并且 MAD2B 的耗竭导致染色体错位的百分比明显增加，并且在有丝分裂过程中 CLTA 重新分布。
