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.

几条证据表明，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。冈田酸是由鞭毛藻产生的毒性多脂肪酸，是蛋白质磷酸酶1型（PP1）和2A型（PP2A）的潜在抑制剂，可去磷酸化丝氨酸丝氨酸和三分之一的真实性细胞中的残留物。冈田酸和钙蛋白A诱导包括SAOS-2，MG63和MCETE-E1细胞在内的成骨细胞中凋亡。 cDNA克隆揭示了大鼠中PP1催化亚基的四种同工型，称为PP1α，PP1γ1，PP1γ2和PP1δ。 PP1靶向亚基被认为本地化为特定的亚细胞成分，并在这些位点调节酶的活性。核苷是一种绝对表达的核仁磷蛋白，主要位于核仁中。 MG63细胞中核素的染色模式与PP1δ的染色模式相似。双荧光图像表明，PP1δ和核素在核olus中代表相同的定位。抗核蛋白抗体与用PP1Δ抗体免疫沉淀的100 kDa蛋白相互作用。但是，抗核蛋白抗体与正常兔血清沉淀的样品没有相互作用。通过Lambda磷酸酶将100 kDa蛋白脱磷酸化为98 kDa蛋白。在放线霉素D处理的细胞中，改变了PP1δ和核素的亚细胞定位。 PP1δ的量增加，而核仁蛋白的去磷酸化形式的水平增加。这些结果表明，PP1δ与核仁蛋白直接相关以脱磷酸化，并参与R-RNA合成。