Hirai M, Amagai M, Tajima S, Nishikawa T, Shimizu N
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Cell Struct Funct. 1988 Dec;13(6):471-80. doi: 10.1247/csf.13.471.
DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.
来自一名沃纳综合征患者的WF-1成纤维细胞的DNA合成受到胎牛血清和成人血清的刺激，但不受包括表皮生长因子、血小板衍生生长因子（PDGF）、成纤维细胞生长因子、胰岛素和12-O-十四烷酰佛波醇-13-乙酸酯（TPA）在内的各种有丝分裂原的刺激。为了阐明对这些有丝分裂原无反应的原因，我们比较了正常成纤维细胞HF-O和沃纳综合征的WF-1细胞中的蛋白质磷酸化速率。PDGF和TPA增强了Mr 80K蛋白的磷酸化，已知该蛋白是蛋白激酶C的底物，在HF-O和WF-1细胞中均如此。这表明涉及PDGF受体、磷脂酰肌醇周转和蛋白激酶C激活的途径在WF-1细胞中起作用。使用抗磷酸酪氨酸抗体通过免疫沉淀在正常HF-O细胞中检测到了几种Mr 250K、135K、110K、78K和42K的磷酸化蛋白。在传代6的沃纳综合征的WF-1细胞中检测到了相同种类的磷酸化蛋白，但仅在用各种有丝分裂原处理时才检测到，并且在传代10的WF-1细胞中，即使经过PDGF或TPA处理也未检测到。这些结果表明，这些靶蛋白磷酸化的减少可能部分导致了沃纳综合征成纤维细胞有丝分裂反应性的降低。
