Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes.
Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes.
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DOI：
10.1091/mbc.e13-10-0603
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发表时间：
2014-03
影响因子：
3.3
通讯作者：
Traktman P
中科院分区：
文献类型：
--
作者：
Molitor TP;Traktman P
VRK 1蛋白激酶调节BAF的磷酸化，BAF与dsDNA和含LEM结构域的蛋白结合。VRK 1缺失增加了BAF在核外围的不动部分，并扰乱了核膜结构。当细胞进入并通过有丝分裂时，它还导致BAF保留在染色体上。自整合屏障因子（BAF）是由BANF 1基因编码的，与核周围的双链DNA和含LEM结构域的蛋白质高亲和力结合。最近，一种BANF 1突变与一种新的人类早衰综合征有关，这些患者的细胞具有异常的核膜。已知BAF与其DNA和蛋白质结合伙伴的相互作用受磷酸化调节，并且先前我们验证了BAF作为VRK 1蛋白激酶的高效底物。在这里，我们表明，在MCF 10a和MDA-MB-231细胞中VRK 1的缺失导致异常的核结构。绿色荧光蛋白（GFP）-BAF在核被膜（NE）上的固定部分升高，表明BAF与其结合伙伴的长期相互作用可能是导致异常NE结构的原因。由于BAF从其结合伙伴的脱离与NE分解相关，我们对对照和VRK 1耗尽的细胞进行了实时成像分析，以可视化有丝分裂期间GFP-BAF的动力学。在缺乏VRK 1的情况下，BAF不会分散，而是从有丝分裂开始就保持染色体结合。VRK 1缺失也增加了后期桥和多极纺锤体的数量。因此，VRK 1对BAF的磷酸化对于正常NE结构和有丝分裂期间BAF-染色体相互作用的适当动力学都是必需的。这些结果与先前在秀丽隐杆线虫和黑腹果蝇中VRK/BAF信号轴的研究一致，并验证VRK 1作为哺乳动物细胞中NE结构和有丝分裂染色体动力学的关键调节因子。
The VRK1 protein kinase regulates the phosphorylation of BAF, which binds to dsDNA and LEM domain–containing proteins. VRK1 depletion increases the immobile fraction of BAF at the nuclear periphery and disturbs nuclear envelope architecture. It also leads to the retention of BAF on chromosomes as cells enter and progress through mitosis. Barrier to autointegration factor (BAF), which is encoded by the BANF1 gene, binds with high-affinity to double-stranded DNA and LEM domain–containing proteins at the nuclear periphery. A BANF1 mutation has recently been associated with a novel human progeria syndrome, and cells from these patients have aberrant nuclear envelopes. The interactions of BAF with its DNA- and protein-binding partners are known to be regulated by phosphorylation, and previously we validated BAF as a highly efficient substrate for the VRK1 protein kinase. Here we show that depletion of VRK1 in MCF10a and MDA-MB-231 cells results in aberrant nuclear architecture. The immobile fraction of green fluorescent protein (GFP)–BAF at the nuclear envelope (NE) is elevated, suggesting that prolonged interactions of BAF with its binding partners is likely responsible for the aberrant NE architecture. Because detachment of BAF from its binding partners is associated with NE disassembly, we performed live-imaging analysis of control and VRK1-depleted cells to visualize GFP-BAF dynamics during mitosis. In the absence of VRK1, BAF does not disperse but instead remains chromosome bound from the onset of mitosis. VRK1 depletion also increases the number of anaphase bridges and multipolar spindles. Thus phosphorylation of BAF by VRK1 is essential both for normal NE architecture and proper dynamics of BAF–chromosome interactions during mitosis. These results are consistent with previous studies of the VRK/BAF signaling axis in Caenorhabditis elegans and Drosophila melanogaster and validate VRK1 as a key regulator of NE architecture and mitotic chromosome dynamics in mammalian cells.