Pim-1 associates with protein complexes necessary for mitosis
Pim-1 associates with protein complexes necessary for mitosis
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DOI：
10.1007/s00412-002-0192-6
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发表时间：
2002-07-01
期刊：
影响因子：
1.6
通讯作者：
Magnuson, NS
中科院分区：
文献类型：
--
作者：
Bhattacharya, N;Wang, ZP;Magnuson, NS
原癌基因Pim-1是一种丝氨酸/苏氨酸激酶，其过表达促进淋巴瘤的形成。Pim-1的正常功能和该基因介导的肿瘤发生的生化机制尚未阐明，尽管最近的研究提供了令人信服的证据表明Pim-1参与了细胞的分化和生存。我们现在提供了Pim-1可能参与增殖过程的第一个证据。通过共聚焦显微镜，我们观察到Pim-1在细胞周期中的动态重新分布，在有丝分裂过程中，蛋白质从间期的核和细胞质移动到纺锤体极。通过计算机搜索位于纺锤体两极的Pim-1的假定底物，我们发现核有丝分裂装置(NUMA)蛋白有两个肽序列，其中包含Pim-1激酶的优先磷酸化位点。重组谷胱甘肽-S-转移酶-PIM-1也容易使免疫沉淀的NUMA磷酸化。通过共聚焦显微镜和免疫共沉淀，我们显示了Pim-1和NUMA蛋白在HeLa细胞中的相互作用，这些蛋白在有丝分裂过程中被诺可达唑阻止。PIM-1似乎还与异染色质相关蛋白1beta(HPIP)以及细胞质蛋白dynein和dynactin通过途径相互作用。与努马形成复杂的队形。在我们的研究中发现，在有丝分裂过程中，过表达的野生型Pim-1-GFP(绿色荧光蛋白)融合蛋白与NUMA共定位于纺锤体极。相反，‘KINK-DEAD’MUT-PIM-1-GFP融合蛋白没有与NUMA共定位，而且似乎促进了细胞凋亡。凋亡细胞死亡的进一步证据是观察到染色体的起泡和碎裂，以及通过共聚焦显微镜检测到的NUMA蛋白水平的下降。这些结果有力地表明，Pim-1激酶很可能通过磷酸化在促进nuA、HPIP、dynein和dynactin之间形成复合体方面发挥作用，Dynein是有丝分裂所必需的复合体。
The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HPIP) and the cytoplasmic proteins dynein and dynactin via. complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HPIP, dynein and dynactin, a complex that is necessary for mitosis.