Gobert S, Duprez V, Lacombe C, Gisselbrecht S, Mayeux P
Institut Cochin de Génétique Moléculaire (ICGM), Institut de la Santé et de la Recherche Médicale (INSERM U363), Université René Descartes, Paris, France.
Eur J Biochem. 1995 Nov 15;234(1):75-83. doi: 10.1111/j.1432-1033.1995.075_c.x.
The survival and proliferation of the UT-7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte-macrophage colony-stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin-stimulated UT-7 cells by anion-exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50-kDa kinase, whereas the second peak was only present in erythropoietin-stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin-stimulated cells. The 44-kDa and 42-kDa forms corresponded to extracellular signal-regulated kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showing that the 45-kDa form is not a shifted form of ERK-1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK-4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin-stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin-induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF-1, was hyperphosphorylated in erythropoietin-stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT-7 erythropoietin-stimulated cells and associated strongly with a unidentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both unstimulated and erythropoietin-stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.
UT-7人白血病细胞系的存活和增殖严格依赖于白细胞介素3、粒细胞巨噬细胞集落刺激因子或促红细胞生成素的存在。在这些细胞中，促红细胞生成素刺激导致几种蛋白质迅速磷酸化，包括促红细胞生成素受体和分子量约为45 kDa的蛋白质，这些蛋白质可能是丝裂原活化蛋白（MAP）激酶。通过阴离子交换色谱法将静止或促红细胞生成素刺激的UT-7细胞的胞质溶胶分离，显示出髓鞘碱性蛋白激酶活性的两个峰。第一个峰的激酶活性与细胞的促红细胞生成素处理无关，对应于一种未鉴定的50 kDa激酶，而第二个峰仅存在于促红细胞生成素刺激的细胞中，对应于分子量为45、44和42 kDa的三种形式的MAP激酶。这三种形式通过疏水色谱法分离，并显示在促红细胞生成素刺激的细胞中被激活。44 kDa和42 kDa的形式分别对应于细胞外信号调节激酶（ERK）-1和ERK-2。有证据表明，45 kDa的形式不是ERK-1的迁移形式，而是对应于一种定义不太明确的MAP激酶形式，可能是先前描述的ERK-4。促红细胞生成素刺激1分钟后检测到MAP激酶激活，1小时以上仍可检测到。促红细胞生成素诱导的MAP激酶刺激和细胞增殖的同时抑制表明MAP激酶激活在促红细胞生成素刺激的细胞增殖中起作用。MAP激酶的潜在激活剂RAF-1在促红细胞生成素刺激的细胞中过度磷酸化，其自身磷酸化活性强烈增加。蛋白衔接子Shc在UT-7促红细胞生成素刺激的细胞中大量磷酸化，并与一种未鉴定的145 kDa蛋白强烈结合。然而，Shc与活化的促红细胞生成素受体结合不佳，并且大多数Shc蛋白在未刺激和促红细胞生成素刺激的细胞中均位于胞质溶胶中。相比之下，Grb2与活化的促红细胞生成素受体有效结合，并且在促红细胞生成素刺激后，相当一部分Grb2与颗粒状亚细胞组分相关联。
