PROTEIN-KINASE-M, THE CYTOSOLIC COUNTERPART OF PROTEIN-KINASE-C, REMODELS THE INTERNAL CYTOSKELETON OF THE MAMMALIAN EGG DURING ACTIVATION
PROTEIN-KINASE-M, THE CYTOSOLIC COUNTERPART OF PROTEIN-KINASE-C, REMODELS THE INTERNAL CYTOSKELETON OF THE MAMMALIAN EGG DURING ACTIVATION
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DOI：
10.1006/dbio.1995.1043
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发表时间：
1995-02-01
影响因子：
2.7
通讯作者：
CAPCO, DG
中科院分区：
文献类型：
--
作者：
GALLICANO, GI;MCGAUGHEY, RW;CAPCO, DG
我们已经研究了细胞内信号在哺乳动物卵子转化为受精卵时重组细胞骨架空间组织的机制。四种不同的方法（一种细胞学方法，两种生化方法和一种药理学方法）表明，蛋白激酶C （PKC）及其细胞质活性对偶物PKM在卵子激活时连续起作用。PKM用于重塑内部细胞骨架。细胞学方法使用PKC报告染料Rim-1绘制了激酶随时间的分布，这表明激酶分布的时间变化，从其在质膜上的初始激活位点到随后与被称为片的中间丝的交联网络相关联。两种生化分析中的第一种，Western blot分析表明，用钙离子载体活化的鸡蛋在洗涤剂可溶性部分含有PKC，在富含薄片的部分含有PKM。在激活鸡蛋之前，在薄片富集的部分中未检测到PKM；在这些鸡蛋的洗涤剂可溶性部分中只检测到PKC。第二个生化分析表明，通过对已知PKC底物的[P-32]ATP标记，PKC响应于钙离子载体激活卵子而激活产生的PKM是一种活性激酶，位于富含薄片的部分。此外，纯化形式的PKM，而不是PKC，可以被证明作用于内部细胞骨架，当灌注到一个渗透的卵子系统。药理学治疗表明[Ca2+](i)的升高不会直接改变卵子的内部细胞骨架。我们的研究结果表明，这种激酶在受精时被用来提供一个内部计时器，首先在细胞外围作为PKC，随后在细胞内部作为PKM。(C) 1995学术出版社，Inc。
We have investigated mechanisms by which intracellular signals act to restructure the spatial organization of the cytoskeleton as the mammalian egg is converted into the zygote. Four distinct approaches (one cytological, two biochemical, and one pharmacological) demonstrate protein kinase C (PKC) and its cytosolic active counterpart, PKM, act in succession at the time of egg activation. PKM serves to remodel the internal cytoskeleton. The cytological approach mapped the distribution of kinase over time using the PKC reporter dye, Rim-1, which demonstrated a temporal shift in kinase distribution from its initial site of activation at the plasma membrane to its subsequent association with a cross-linked network of intermediate filaments referred to as sheets. The first of two biochemical analyses, Western blot analysis, demonstrated that eggs activated with calcium ionophore contained PKC in the detergent-soluble fraction and PKM in the sheet enriched fraction. Prior to egg activation, PKM is not detected in the sheet enriched fraction; only PKC is detected in the detergent-soluble fraction of these eggs. The second biochemical analysis demonstrated, via [P-32]ATP labeling of a known PKC substrate, that the PKM generated from activation of PKC in response to activation of eggs with calcium ionophore is an active kinase and is located in the sheet-enriched fraction. In addition, purified forms of PKM, but not PKC, could be shown to act on the internal cytoskeleton when perfused into a permeabilized egg system. Pharmacological treatments demonstrate that elevation of [Ca2+](i) does not act directly to alter the internal cytoskeleton of the egg. Our results suggest that this kinase is employed at the time of fertilization to provide an internal chronometer acting first at the cell periphery as PKC and subsequently in the cell interior as PKM. (C) 1995 Academic Press, Inc.