Walker G R, Shuster C B, Burgess D R
Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.
J Cell Sci. 1997 Jun;110 ( Pt 12):1373-86. doi: 10.1242/jcs.110.12.1373.
Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.
过去几年的研究已经证明了蛋白质磷酸化在调节有丝分裂和细胞周期中的核心作用。然而，对于调节进入有丝分裂的机制如何促进胞质分裂期间形成的基于肌动球蛋白的收缩环的位置和时间调节，我们知之甚少。最近的研究表明p34cdc2是肌球蛋白II活性的负调节因子，这表明有丝分裂周期和胞质分裂之间存在联系。为了研究蛋白质磷酸化与胞质分裂之间的关系，我们在海胆卵皮质的分离模型中检测了肌动蛋白相关皮质细胞骨架（CSK）蛋白的体内和体外磷酸化。对用32P-正磷酸盐标记的卵或受精卵衍生的皮质进行检测，发现了许多与皮质相关的磷酸化蛋白，包括20、43和66 kDa的多肽。当分离的皮质在体外与[32P]ATP孵育时，也检测到这三种主要的磷蛋白，这表明磷酸化这些底物的激酶也与皮质特异性相关。体内和体外的激酶活性受受精刺激，并表现出细胞周期依赖性活性。凝胶自磷酸化分析、激酶分析和免疫印迹分析揭示了p34cdc2以及丝裂原活化蛋白激酶家族成员的存在，其在CSK中的活性在细胞分裂时达到峰值。诺考达唑可抑制微管形成，从而阻断胞质分裂，显著延迟皮质蛋白磷酸化峰值时间以及全细胞组蛋白H1激酶活性峰值时间。这些结果表明，调节胞质分裂期间皮质收缩的一个关键因素是与皮质细胞骨架相关的蛋白激酶活性的时间，而这反过来又受有丝分裂装置的调节。
