A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase.
A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase.
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DOI：
10.1083/jcb.119.4.843
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发表时间：
1992-11
期刊：
影响因子：
--
通讯作者：
Sloboda RD
中科院分区：
文献类型：
--
作者：
Johnston JA;Sloboda RD
62 kD的蛋白质是钙/钙调蛋白依赖性蛋白激酶的底物，并且两种蛋白质与分离的有丝分裂装置共纯化（Dinsmore，J.H.，和R. D.斯洛博达。1988. Cell. 53：769- 780）。磷酸化的62-kD蛋白质增加受精后，磷酸盐的最大掺入发生在中期后期和后期，并直接与微管拆卸通过体外实验与分离的有丝分裂装置确定。因为62-kD蛋白磷酸化以类似于有丝分裂细胞周期蛋白积累的模式发生，所以进行实验以确定细胞周期蛋白和62- kD蛋白之间的关系。用[35 S]蛋氨酸连续标记海洋胚胎，以及使用特异性抗体的海洋胚胎蛋白的免疫印迹，用于识别细胞周期蛋白和62-kD蛋白。这些结果清楚地表明，62-kD蛋白是不同的细胞周期蛋白，不像细胞周期蛋白，是一个恒定的成员的细胞蛋白池在前两个细胞周期的海胆和冲浪蛤胚胎。用免疫荧光显微镜观察完整的卵子和胚胎也得到了类似的结果。此外，免疫金电子显微镜显示，62-kD蛋白与微管的有丝分裂器在分裂细胞。有趣的是，在细胞周期中，该蛋白质改变其亚细胞分布相对于微管。具体来说，在有丝分裂过程中，62- kD蛋白与有丝分裂器相关联;然而，在核膜破裂之前，62-kD蛋白被限制在细胞核中。分裂后期后，62-kD蛋白返回细胞核，在那里它驻留，直到下一个细胞周期的核膜解体。
A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769- 780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62- kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62- kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.