Mahadevan L C, Wills A J, Hirst E A, Rathjen P D, Heath J K
Department of Biochemistry, Oxford University.
Oncogene. 1990 Mar;5(3):327-35.
Epidermal Growth Factor (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 fibroblasts by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a 15 kDa chromatin-associated phosphoprotein (pp15), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription factors that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.
表皮生长因子（EGF）和十四酰佛波醇乙酸酯（TPA）主要通过分别激活不同的蛋白激酶——EGF受体酪氨酸激酶和蛋白激酶C，在C3H 10T1/2成纤维细胞中引发信号级联反应；在信号起始阶段不存在信号交叉。然而，我们在此表明，这两种因子都会迅速引发共同的细胞内反应，包括一种33 kDa磷蛋白（pp33）的复合形式和与染色质相关形式的磷酸化、一种15 kDa与染色质相关的磷蛋白（pp15）的磷酸化，以及包括c-fos原癌基因在内的一组共同基因的转录激活。2-氨基嘌呤能特异性消除对EGF和TPA作出反应时复合形式和与染色质相关的pp33磷酸化，以及这两种因子对c-fos的诱导。蛋白激酶C的激活以及刺激细胞中与血清反应元件（SRE）、TPA反应元件（TRE）或NFkB位点结合的转录因子水平相对不受2-氨基嘌呤的影响。据我们所知，这是首次证明，通过使用能选择性阻断TPA刺激的pp33磷酸化的2-氨基嘌呤，尽管蛋白激酶C仍保持完全活性，但仍可阻断TPA处理细胞中c-fos的诱导。此外，我们在此表明，尽管2-氨基嘌呤消除了EGF和TPA刺激的c-fos诱导，但刺激细胞的核提取物中TRE结合活性的出现不受影响，这表明EGF和TPA刺激的TRE结合活性诱导利用的是现有蛋白质，并不依赖于新的c-FOS合成。这些结果表明，2-氨基嘌呤敏感的复合形式和与染色质相关的pp33磷酸化可能对响应EGF和TPA时的c-fos诱导至关重要。
