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首页> 外文OA文献 >The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.
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The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

机译：蛋白激酶激活事件在级联中调节定时，该级联调节紫锥花雄蕊毛细胞的有丝分裂进程。

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摘要

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.
机译：紫草植物Tradescantia virginiana的雄蕊毛细胞通过有丝分裂表现出异常可预测的进展速度，特别是从核被膜破裂（NEBD）到细胞分裂开始之时。通过前中期和中期的可预测进展速度已使这些细胞成为确定触发进入后期的调节事件时间的有用模型系统。大量研究表明，提高一种或多种蛋白激酶活性是进入后期的必要先决条件。当前的实验采用两种策略来测试这些蛋白激酶活性的升高何时真正发生在中期。在灌注过程中，我们在前中期或中期将蛋白激酶抑制剂K-252a，星形孢菌素或钙磷蛋白C在已知时间间隔10分钟添加到活的雄蕊毛细胞中，并监测随后的进展到后期的速率。中期转运时间根据向细胞中添加K-252a或星形孢菌素的时间而改变；在前中期开始的中期至早期中期的治疗大大延长了中期的转运时间。在中期中期开始治疗后，NEBD后约15至21分钟，转运时间正常。 Calphostin C对中期的过渡时间没有重大影响。并行地，在前中期和中期的预定时间点，向细胞显微注射已知量的通用蛋白激酶底物肽VRKRTLRRL。在细胞溶质浓度为100 nM至1 microM时，当在NEBD之后的前4分钟内（在NEBD之后的7.5至9分钟内）的任何时间点将肽注入有丝分裂细胞的胞质溶胶中时，该肽的中期转运时间是原来的两倍或三倍NEBD后14至16分钟，NEBD后21分钟或NEBD后24分钟。在所使用的浓度下以及在这些短暂的间隔内，当蛋白激酶激活发生或当内源底物水平接近该肽水平时，该肽似乎起竞争抑制剂的作用，以揭示拐点。这些拐点的时机与前中期和中期的蛋白激酶活性变化相吻合，正如我们用广谱激酶抑制剂对细胞的灌注所表明的那样。总的来说，我们的结果表明，在后期达到顶峰的级联是复杂的，并且涉及几个连续的蛋白激酶激活步骤，这些步骤被中期中期的一种或多种蛋白磷酸酶的激活所打断。

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    Wolniak, S M; Larsen, P M;

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  • 年度 1995
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