Importance of tyrosine phosphatases in the effects of cell-cell contact and microenvironments on EGF-stimulated tyrosine phosphorylation.
Importance of tyrosine phosphatases in the effects of cell-cell contact and microenvironments on EGF-stimulated tyrosine phosphorylation.
复制标题
酪氨酸磷酸酶在细胞间接触和微环境对 EGF 刺激的酪氨酸磷酸化的影响中的重要性。
DOI：
10.1002/jcp.1041510302
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发表时间：
1992
影响因子：
5.6
通讯作者：
Sutherland,RM
中科院分区：
文献类型：
--
作者：
Mansbridge,JN;Knuchel,R;Knapp,AM;Sutherland,RM
我们比较了A431细胞在各种细胞密度下单层生长时的EGF反应，以研究细胞接触和三维结构对信号转导的影响。A431鳞状细胞癌细胞株生长在我们的实验室是不受EGF的单层培养生长。然而，作为三维多细胞球体，生长受到EGF的刺激。在EGF存在下可达到的最大体积是在其不存在下的30倍以上。在这些条件下，通过免疫组织化学和蛋白质印迹法比较EGF依赖的酪氨酸磷酸化。在使用已发表程序的初始实验中，酪氨酸磷酸化在单层中具有密度依赖性，在球状体中无法检测到。然而，通过添加高浓度的蛋白酪氨酸磷酸酶抑制剂（1 mM Zn++和VO 43 −）消除了密度依赖性。因此，单层细胞中EGF刺激的酪氨酸磷酸化的密度依赖性在很大程度上是磷酸酶活性变化的结果，而不是激酶。使用高浓度的磷酸酶抑制剂，磷酸酪氨酸是清楚可见的免疫组织化学在球体的最外层的细胞，但它仍然是不可见的球体中心。球体内缺乏反应与EGF受体的存在或EGF的扩散无关。在配套实验中，我们发现EGF受体的染色均匀地存在于整个球体中，并且在实验条件下EGF渗透到其中心。因此，虽然酪氨酸磷酸酶活性的增加是影响外细胞中酪氨酸磷酸化的主要因素，但其他因素在内细胞中也很重要。我们的结论是，酪氨酸磷酸酶活性的增加是最重要的组成部分的EGF信号转导系统的适应高细胞密度在单层培养。在球状体中，酪氨酸磷酸酶也增强，但其他因素，如TGF-α的自分泌合成，以及可能的EGF受体和细胞形状的细胞分布，发挥作用。© 1992 Wiley利斯公司
We have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities of as multicellular spheroids in order to investigate the effects of cell contact and 3‐dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3‐dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF‐dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density‐dependent in monolayers and undetectable in spheroids. However, the density‐dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++and VO43−). The density dependence of EGF‐stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF‐α and possibly the cellular distribution of EGF receptors and cell shape, play a role. © 1992 Wiley‐Liss, Inc.