Besterman B, Schultz R M
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
J Exp Zool. 1990 Oct;256(1):44-53. doi: 10.1002/jez.1402560107.
We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.
我们研究了酪氨酸蛋白磷酸化抑制剂染料木黄酮对小鼠单细胞胚胎的影响，因为在体细胞中，有丝分裂刺激引发的酪氨酸蛋白磷酸化与DNA合成的起始有关。染料木黄酮以浓度依赖且可逆的方式抑制单细胞胚胎的分裂；而生物chanin A作为酪氨酸蛋白磷酸化的较弱抑制剂，对细胞分裂的抑制作用也较弱。染料木黄酮不抑制[35S]甲硫氨酸掺入，但抑制[3H]胸苷掺入。与染料木黄酮通过抑制DNA合成来抑制分裂的能力一致的是，其抑制分裂能力的丧失与单细胞胚胎退出S期相对应。染料木黄酮可能在原位抑制酪氨酸蛋白磷酸化，因为它使单细胞胚胎中[32P]磷酸酪氨酸的相对含量降低了80%；染料木黄酮既不抑制[32P]正磷酸盐摄取也不抑制其掺入。正如预期的那样，染料木黄酮对抑制第一个细胞周期中磷蛋白合成模式的变化影响很小，因为酪氨酸蛋白磷酸化在总蛋白磷酸化中所占比例很小。对转移到Immobilon上的[32P]放射性标记磷蛋白进行碱处理，发现一组Mr = 32,000的耐碱磷蛋白，其磷酸化呈现细胞周期变化。尽管这些特性表明这些磷蛋白可能与p34cdc2蛋白激酶有关，但对[32P]放射性标记磷蛋白的磷酸氨基酸分析表明，它们的磷酸酪氨酸并不富集；无活性的p34cdc2蛋白激酶含有高水平的磷酸酪氨酸。这些实验结果表明，对受精精子产生反应的酪氨酸蛋白磷酸化可能参与启动单细胞胚胎中的DNA合成，以及将减数分裂细胞周期转变为有丝分裂细胞周期。
