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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.

一些证据表明，蛋白质磷酸化和去磷酸化已被认为是各种组织中细胞增殖、分化和凋亡的关键机制。冈田酸是由甲藻产生的有毒聚醚脂肪酸，并且是蛋白磷酸酶1型（PP 1）和2A型（PP 2A）的有效抑制剂，其使真核细胞中的丝氨酸和苏氨酸残基去磷酸化。冈田酸和calyculin A诱导成骨细胞包括Saos-2、MG 63和MCeTe-E1细胞凋亡。cDNA克隆显示大鼠存在4种PP 1催化亚基的异构体，分别命名为PP 1 α、PP 1 γ1、PP 1 γ2和PP 1 δ。PP 1靶向亚基被认为定位于特定的亚细胞组分并调节这些位点的酶活性。核仁素是一种高表达的核仁磷蛋白，主要定位于核仁中。核仁素在MG 63细胞中的染色模式与PP 1 δ相似。双重荧光图像显示，PP 1 δ和核仁素在核仁中具有相同的定位。抗核仁素抗体与PP 1 δ抗体免疫沉淀的100 kDa蛋白相互作用。但抗核仁素抗体与正常兔血清沉淀的样品不发生相互作用。100 kDa的蛋白被λ磷酸酶脱磷酸化为98 kDa的蛋白。在放线菌素D处理的细胞中，PP 1 δ和核仁素的亚细胞定位发生变化。PP 1 δ的量增加，而核仁素的去磷酸化形式的水平增加。这些结果表明，PP 1 δ与核仁素直接结合，使该蛋白脱磷酸化，并参与r-RNA的合成。