MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases.
MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases.
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MPM-2 抗体反应性磷酸化可通过动粒结合的可溶性激酶在去污剂提取的细胞中产生。
作者：
L. Renzi;M. Gersch;M. S. Campbell;L. Wu;S. Osmani;G. Gorbsky
MPM-2抗体标记有丝分裂特异的和细胞周期调节的磷酸蛋白。MPM-2抗体识别的有丝分裂染色体的主要磷蛋白是DNA拓扑异构酶II(TopoII)α和β。在对PtK1细胞骨架的免疫荧光研究中，MPM-2抗体标记了在有丝分裂细胞的动心、染色体臂、中体和纺锤体极发现的磷蛋白。在没有磷酸酶抑制剂的细胞中，运动中心的MPM-2抗体的标记大大减少。然而，在细胞骨架中，这个表位可以通过稳定结合在动粒上的激酶的作用而再生。为了确定引起这些磷酸化的内源性激酶的特征，测试了各种不同的激酶抑制剂。我们发现MPM-2表位在广泛特异性的激酶抑制剂K-252a、星形孢子素和2-氨基嘌呤的存在下不会重新磷酸化。其他几种抑制剂对再磷酸化没有影响，表明在动点的内源性MPM-2激酶不是p34cdc2、酪蛋白激酶II、MAP激酶、蛋白激酶A或蛋白激酶C。加入N-乙基马来酰亚胺使内源动粒激酶失活；这使得可以在动粒再磷酸化试验中检测几种纯化的激酶。活化的p34cdc2-Cyclin B、酪蛋白激酶II和MAP激酶不能产生MPM-2的磷酸表位。然而，曲霉菌表达的NIMA和超速速有丝分裂HeLa细胞提取物能够催化MPM-2表位在运动中心的重新磷酸化。此外，有丝分裂HeLa细胞提取物的分离表明，在动粒和染色体臂上创建MPM-2表位的激酶是不同的。我们的结果表明，多种激酶(无论是可溶的还是动粒结合的)，包括哺乳动物NIMA的同源物，都可以创建MPM-2磷酸表位。动粒结合的激酶催化MPM-2磷酸表位的形成，可能在有丝分裂后期的有丝分裂动粒组装和姐妹染色单体分离等关键事件中发挥重要作用。
The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.
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DOI：
10.1073/pnas.80.10.2926
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DAVIS, FM;TSAO, TY;RAO, PN
通讯作者：
RAO, PN
NIMA 蛋白激酶在 p34cdc2/cyclin B 下游过度磷酸化并被激活：两个有丝分裂促进激酶的协调。
DOI：
10.1002/j.1460-2075.1995.tb07079.x
发表时间：
1995
期刊：
The EMBO journal
影响因子：
--
作者：
Ye,XS;Xu,G;Pu,RT;Fincher,RR;McGuire,SL;Osmani,AH;Osmani,SA
通讯作者：
Osmani,SA
DOI：
10.1073/pnas.86.13.4982
发表时间：
1989-07-01
影响因子：
11.1
作者：
KUANG, J;ZHAO, JY;RAO, PN
通讯作者：
RAO, PN
磷蛋白是有丝分裂微管组织中心的组成部分。
DOI：
10.1073/pnas.81.14.4439
发表时间：
1984
影响因子：
11.1
作者：
Vandre,DD;Davis,FM;Rao,PN;Borisy,GG
通讯作者：
Borisy,GG
使用 TALE 灯照亮果蝇发育过程中的 DNA 复制。
DOI：
10.1016/j.cub.2014.01.023
发表时间：
2014
期刊：
Current biology : CB
影响因子：
--
作者：
Yuan,Kai;Shermoen,AntonyW;O'Farrell,PatrickH
通讯作者：
O'Farrell,PatrickH