Patel Jennifer T, Bottrill Andrew, Prosser Suzanna L, Jayaraman Sangeetha, Straatman Kees, Fry Andrew M, Shackleton Sue
a Department of Biochemistry; University of Leicester; Leicester, UK.
Nucleus. 2014 Sep-Oct;5(5):462-73. doi: 10.4161/nucl.36232.
At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.
在高等真核生物有丝分裂开始时，核膜（NE）会经历剧烈解构，以允许复制后的染色体分离。研究表明，在核膜破裂（NEBD）过程中，核纤层广泛的蛋白质网络通过核纤层蛋白和几种内核膜（INM）蛋白的磷酸化而被拆解。由SUN和nesprin蛋白组成的LINC复合物参与了核膜处的多种相互作用，并通过将细胞核与细胞骨架相连，在细胞核和细胞力学中发挥着至关重要的作用。在此，我们表明，位于内核膜的SUN1在其核质N端的至少3个位点上发生有丝分裂特异性磷酸化。我们进一步确定Cdk1是负责丝氨酸48和333磷酸化的激酶，而丝氨酸138则由Plk1磷酸化。在有丝分裂细胞中，SUN1失去了与N端结构域结合伙伴核纤层蛋白A/C、emerin和短nesprin-2异构体的相互作用。此外，一种三磷酸模拟SUN1突变体显示出溶解度增加，并且在核膜处的滞留减少。相比之下，SUN1 C端与nesprin-2的KASH结构域之间的核心LINC复合物相互作用在有丝分裂期间得以维持。这些数据共同支持了一个模型，即SUN1的有丝分裂磷酸化破坏了与核质结合伙伴的相互作用，促进了核纤层的拆解以及潜在的染色质相互作用。同时，我们的数据进一步表明，核心LINC复合物在核膜破裂中发挥着积极作用。
