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首页> 外文期刊>Biology Open >Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly
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Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

机译：有丝分裂高尔基体拆卸过程中GRASP65的顺序磷酸化。

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GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.
机译：在细胞周期中，高尔基体的拆卸和重新组装需要有丝分裂期间的GRASP65磷酸化和有丝分裂之后的去磷酸化。已经确定了GRASP65上至少有8个磷酸化位点，但是至今尚不清楚它们是否在有丝分裂过程中以协同方式被修饰。在这项研究中，我们提出了磷酸特异性抗体，它们分别识别GRASP65的磷酸化T220 / T224，S277和S376残基。生化分析表明，cdc2使所有三个位点都磷酸化，而plk1增强了磷酸化。使用这些抗体进行双重和三次标记的显微镜研究表明，在细胞周期中，磷酸化和去磷酸化依次发生。当新的高尔基体重新组装时，S277和S376从G2晚期阶段到中期直至末期被磷酸化。 T220 / 224直到前期才进行修改，但从前中期到后期都进行了高度修改。在中期，磷酸化-T220 / 224信号既定位于高尔基霾和有丝分裂的高尔基体簇上，分别代表分散的高尔基囊泡和高尔基残体，而磷酸化S277和S376标记则更集中于有丝分裂的高尔基体上。抗磷酸化的GRASP65突变体T220A / T224A的表达比S277A和S376A突变体的表达在更大程度上抑制了有丝分裂高尔基体片段化。在胞质分裂中，T220 / 224的去磷酸化发生在S277之前，但在S376之后。这项研究提供的证据表明，在有丝分裂期间，GRASP65在不同位点依次被磷酸化和去磷酸化，以协调细胞分裂过程中高尔基体的分解和重新组装，其中T220 / 224位点的磷酸化是最关键的过程。

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