Protein dephosphorylation and apoptosis in osteoblasts

成骨细胞中的蛋白质去磷酸化和细胞凋亡

基本信息

批准号：
11671806
负责人：
HANEJI Tatsuji
金额：
$ 2.24万
依托单位：
University of Tokushima School of Dentistry
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671806/
关键词：
apoptosis Fas antigen Fas ligand nucleolin okadaic acid osteoblast protein phosphatase protein phosphatase calyculin A

项目摘要

Several lines of evidence indicate that protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell proliferation, differentiation, and apoptosis in various tissues. Okadaic acid is a toxic polyether fatty acid produced by dinoflagellates and is a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A) that dephosphorylate serine and threonie residues in eukaryotic cells. Okadaic acid and calyculin A induce apoptosis in osteoblastic cells including Saos-2, MG63 and MCeTe-E1 cells. cDNA cloning revealed the existence of four isoforms of PP1 catalytic subunit in rat, termed PP1α, PP1γ1, PP1γ2, and PP1δ. PP1 targeting subunits is thought to localize to specific subcellular component and to modulate the activity of the enzyme at these sites. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in nucleolus. Staining pattern of nucleolin in MG63 cells is similar to that of the PP1δ. The dual fluorescence image revealed that PP1δ and nucleolin represent same localization in nucleolus. The anti-nucleolin antibody interacted with the 100 kDa protein immunoprecipitated with PP1δ antibody. However, anti-nucleolin antibody did not interact with the samples precipitated with the normal rabbit serum. The 100 kDa protein was dephosphorylate into 98 kDa proteins by lambda phosphatase. In the actinomycin D-treated cells, subcellular localization of PP1δ and nucleolin was changed. The amount of PP1δ increased whereas the level of dephosphorylated form of nucleolin increased. These results indicate that PP1δ associate with nucleolin directly to dephosphorylate this protein and is involved in r-RNA synthesis.

多项证据表明，蛋白质磷酸化和去磷酸化已被认为是多种组织中细胞增殖、分化和凋亡的关键机制，冈田酸是甲藻产生的有毒聚醚脂肪酸，是 1 型蛋白磷酸酶的有效抑制剂。 (PP1) 和 2A 型 (PP2A) 使真核细胞中的丝氨酸和苏氨酸残基去磷酸化，冈田酸和花萼蛋白 A 诱导细胞凋亡。包括 Saos-2、MG63 和 MCeTe-E1 细胞在内的成骨细胞的 cDNA 克隆揭示了大鼠体内存在 PP1 催化亚基的四种亚型，称为 PP1α、PP1γ1、PP1γ2，并且 PP1 靶向亚基被认为定位于特定的亚细胞成分。并调节这些位点的酶活性。核仁蛋白是一种丰富表达的核仁。 MG63 细胞中核仁素的染色模式与 PP1δ 相似。双荧光图像显示 PP1δ 和核仁素在核仁中具有相同的定位。抗核仁素抗体与免疫沉淀的 100 kDa 蛋白相互作用。然而，抗核蛋白抗体不与沉淀的样品相互作用。与正常兔血清相比，100 kDa 的蛋白质被 lambda 磷酸酶去磷酸化为 98 kDa 的蛋白质。在放线菌素 D 处理的细胞中，PP1δ 和核仁素的亚细胞定位发生变化，而核仁素的去磷酸化水平增加。这些结果表明 PP1δ 与核仁素直接结合，使该蛋白质去磷酸化，并参与其中。 r-RNA合成。