Taagepera S, Campbell M S, Gorbsky G J
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Exp Cell Res. 1995 Nov;221(1):249-60. doi: 10.1006/excr.1995.1373.
We have detected novel phosphotyrosine epitopes at the kinetochores of mitotic chromosomes in rat kangaroo PtK1 and mouse P388D1 tissue culture cells. Immunofluorescence labeling of detergent-resistant cytoskeletons reveals that these phosphotyrosine epitopes are tightly bound at the centrosomes and kinetochores of mitotic cells. These phosphoepitopes are found at the kinetochores during only prophase and prometaphase. Inclusion of a mixture of phosphatase inhibitors in the cell extraction procedure was necessary to preserve these previously undetected phosphotyrosine epitopes. The use of the phosphatase inhibitor mixture also improved the detection of the centrosome and kinetochore antigens recognized by the monoclonal antibody MPM-2. The MPM-2 antibody labels a subset of phosphothreonine-containing antigens found primarily during M phase. Ultrastructural immunolabeling studies indicated that both the phosphotyrosine and the MPM-2 phosphoepitopes were contained in both the outer and the inner dense plaques of the kinetochore. We developed large-scale chromosome isolation procedures designed to maintain chromosome protein phosphorylation. Immunoblot analysis revealed that the phosphotyrosine and MPM-2 antibodies recognized a number of chromosomal proteins, some of which were concentrated in the chromosome scaffold fraction prepared by nuclease digestion and salt extraction of whole chromosomes. The strictly regulated appearance of the phosphotyrosine and MPM-2 epitopes at the kinetochores of chromosomes during various stages of mitosis suggests that these phosphoepitopes may be involved in signal transduction pathways controlling kinetochore assembly and function during mitosis.
我们在大鼠袋鼠PtK1和小鼠P388D1组织培养细胞的有丝分裂染色体动粒上检测到了新的磷酸酪氨酸表位。对耐去污剂细胞骨架的免疫荧光标记显示，这些磷酸酪氨酸表位紧密结合在有丝分裂细胞的中心体和动粒上。这些磷酸表位仅在前期和前中期出现在动粒上。在细胞提取过程中加入磷酸酶抑制剂混合物对于保留这些先前未检测到的磷酸酪氨酸表位是必要的。磷酸酶抑制剂混合物的使用还改善了单克隆抗体MPM-2所识别的中心体和动粒抗原的检测。MPM-2抗体标记了主要在M期发现的一组含磷酸苏氨酸的抗原。超微结构免疫标记研究表明，磷酸酪氨酸和MPM-2磷酸表位都存在于动粒的外层和内层致密斑中。我们开发了大规模的染色体分离程序，旨在维持染色体蛋白的磷酸化。免疫印迹分析显示，磷酸酪氨酸抗体和MPM-2抗体识别了许多染色体蛋白，其中一些集中在通过核酸酶消化和全染色体盐提取制备的染色体支架部分中。在有丝分裂的各个阶段，染色体动粒上磷酸酪氨酸和MPM-2表位的严格调控出现表明，这些磷酸表位可能参与了控制有丝分裂期间动粒组装和功能的信号转导途径。
