Mahadevan L C, Heath J K, Leichtfried F E, Cumming D V, Hirst E M, Foulkes J G
Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, Mill Hill, London, UK.
Oncogene. 1988 Mar;2(3):249-57.
A sequential extraction procedure has been used to prepare three protein fractions from control and mitogen-stimulated [32P]-PO4-labelled fibroblasts, the first containing soluble and membrane-bound proteins, the second mainly the cytoskeletal proteins vimentin and actin, and the third, a chromatin-derived fraction enriched in histones. We describe here an analysis of the mitogen-stimulated changes in the [32P] labelled phosphoprotein composition of these fractions. The most obvious changes in response to epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and bombesin were the rapid appearance of 33 and 15 kDa phosphoproteins in the chromatin-derived fraction. The epidermal growth factor- and basic fibroblast growth factor-stimulated 33 kDa phosphoprotein produced similar chymotryptic peptides and was phosphorylated on serine residues. DNAse/RNAse treatment of the lysates was essential for the extraction of the 33 kDa phosphoprotein. Further, its presence could be demonstrated in preparations of conventionally purified nuclei. An in situ extraction procedure has been used to provide morphological verification of the sequential extraction data. The final structure containing these phosphoproteins is clearly derived from nuclei, enriched in histones, stains for DNA and appears by electron microscopy, to be homogenously composed of chromatin-like material. Thus, we describe here the rapid mitogen-induced appearance of novel phosphoproteins in the nucleus, raising the possibility that they may be involved in orchestrating early nuclear responses to polypeptide growth factors.
已采用一种连续提取程序，从对照和成丝分裂原刺激的[32P]-磷酸标记的成纤维细胞中制备出三种蛋白质组分，第一种包含可溶性和膜结合蛋白，第二种主要是细胞骨架蛋白波形蛋白和肌动蛋白，第三种是富含组蛋白的染色质衍生组分。我们在此描述了对这些组分中[32P]标记的磷蛋白组成的有丝分裂原刺激变化的分析。对表皮生长因子、胚胎癌衍生生长因子、碱性成纤维细胞生长因子和蛙皮素的反应中，最明显的变化是在染色质衍生组分中迅速出现33 kDa和15 kDa的磷蛋白。表皮生长因子和碱性成纤维细胞生长因子刺激产生的33 kDa磷蛋白产生相似的胰凝乳蛋白酶肽段，并在丝氨酸残基上磷酸化。裂解物的DNA酶/RNA酶处理对于33 kDa磷蛋白的提取至关重要。此外，在常规纯化的细胞核制剂中可以证明其存在。已使用原位提取程序对连续提取数据进行形态学验证。包含这些磷蛋白的最终结构显然源自细胞核，富含组蛋白，对DNA染色，并且通过电子显微镜观察，似乎由类似染色质的物质均匀组成。因此，我们在此描述了有丝分裂原诱导的细胞核中新型磷蛋白的快速出现，这增加了它们可能参与协调对多肽生长因子的早期核反应的可能性。
