Protein dephosphorylation and apoptosis in osteoblasts

一些证据表明，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。冈田酸是一种由鞭毛藻产生的有毒聚醚脂肪酸，是真核细胞中丝氨酸和苏氨酸残基去磷酸化的1型和2A型蛋白磷酸酶的有效抑制剂。冈田酸和calyculin A诱导成骨细胞凋亡，包括Saos-2、MG63和MCeTe-E1细胞。cDNA克隆结果显示，PP1催化亚基在大鼠体内存在PP1α、PP1γ1、PP1γ2和PP1δ四个亚型。PP1靶向亚基被认为定位于特定的亚细胞成分，并在这些位点调节酶的活性。核仁蛋白是一种大量表达的核仁磷酸化蛋白，主要位于核仁中。MG63细胞核仁蛋白的染色模式与PP1δ相似。双荧光图像显示PP1δ和核蛋白在核仁中具有相同的定位。抗核蛋白抗体与PP1δ抗体免疫沉淀的100 kDa蛋白相互作用。而抗核仁蛋白抗体与正常兔血清沉淀的样品无相互作用。100kda蛋白被lambda磷酸酶去磷酸化为98kda蛋白。在放线菌素d处理的细胞中，PP1δ和核蛋白的亚细胞定位发生了变化。PP1δ的数量增加，核蛋白去磷酸化形式的水平增加。这些结果表明PP1δ与核仁蛋白直接结合使该蛋白去磷酸化，并参与r-RNA的合成。

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.