Svetlikova S B, Abramova M V, Kukushkin A N, Darieva Z A, Pospelova T V, Pospelov V A
Institute of Cytology, RAS, St. Petersburg.
Tsitologiia. 2001;43(10):961-8.
Proteins of Ras family play an important role in regulation of cell growth and proliferation, and their mutations can lead to growth factor-independent proliferation due to constitutive activity of various signal transduction cascades. In the present work, we studied the activity of ERK, JNK and p38 MAP-kinase cascades in rat embryo fibroblast cells transformed with oncogenes E1A and cHa-ras. These transformed cells are characterized by a high and non-regulated activity of transcription factor AP-1 involved in the regulation of cell proliferation. Since phosphorylation of AP-1 depends on the activity of relevant MAP-kinase cascades (ERK, JNK and p38), we analysed the expression of non-phosphorylated forms of the kinases and their phosphorylated state in E1A + cHa-ras cells using antibodies specific to non-phosphorylated and phosphorylated proteins. It has been established that transformed cells contain higher amounts of non-phosphorylated ERK, JNK and p38 kinases, thus implying a reduced degradation of these and other proteins in the transformants. The content of phosphorylated (active) forms studied in Western blot-analysis with phosphoantibodies was shown to be also higher in exponentially growing E1A + cHa-ras cells. But serum stimulation of the starved cells gave insignificant rise to an increase of ERK, JNK and p38 phosphorylation. Nevertheless, an in vitro kinase assay performed with the kinases, either immunoprecipitated by antibody or bound to GST-fusion substrates, enabled us to show a certain level of stimulation of c-Jun-associated (JNK) and MEF2A-associated (p38) kinase activity in serum stimulated E1A + cHa-ras cells. Thus, the obtained results show that transformation of fibroblasts with E1A and ras oncogenes may contribute to constitutive activation of ERK, JNK and p38 kinase cascades responsible for a high and non-regulated activity of MAP-kinase-dependent transcription factors, in particular AP-1.
Ras家族蛋白在细胞生长和增殖的调控中发挥着重要作用，其突变可导致各种信号转导级联反应的组成性激活，从而引发不依赖生长因子的增殖。在本研究中，我们研究了用癌基因E1A和cHa-ras转化的大鼠胚胎成纤维细胞中ERK、JNK和p38丝裂原活化蛋白激酶级联反应的活性。这些转化细胞的特征是参与细胞增殖调控的转录因子AP-1具有高活性且不受调控。由于AP-1的磷酸化依赖于相关丝裂原活化蛋白激酶级联反应（ERK、JNK和p38）的活性，我们使用针对非磷酸化和磷酸化蛋白的特异性抗体，分析了E1A + cHa-ras细胞中激酶的非磷酸化形式及其磷酸化状态的表达。已经确定，转化细胞中含有更高量的非磷酸化ERK、JNK和p38激酶，这意味着这些蛋白和其他蛋白在转化体中的降解减少。在用磷酸抗体进行的蛋白质印迹分析中研究的磷酸化（活性）形式的含量在指数生长的E1A + cHa-ras细胞中也更高。但是，饥饿细胞的血清刺激导致ERK、JNK和p38磷酸化的增加不显著。然而，用抗体免疫沉淀或与GST融合底物结合的激酶进行的体外激酶测定，使我们能够显示血清刺激的E1A + cHa-ras细胞中c-Jun相关（JNK）和MEF2A相关（p38）激酶活性有一定程度的刺激。因此，获得的结果表明，用E1A和ras癌基因转化成纤维细胞可能有助于ERK、JNK和p38激酶级联反应的组成性激活，这些级联反应负责丝裂原活化蛋白激酶依赖性转录因子，特别是AP-1的高活性且不受调控的活性。
