Protein phosphatases and cleavage of nucleolin in apoptosis-induced osteoblastic cells

蛋白质的磷酸化和去磷酸化被认为是细胞功能中的一个关键机制。冈田酸是PP1和PP2A的有效抑制剂，可诱导包括成骨细胞在内的人或小鼠细胞的凋亡。核仁是一种大量表达的核仁磷酸蛋白，主要分布在核仁中。在培养的成骨细胞中核仁素的染色模式与Pp1δ相似。用免疫细胞化学和免疫沉淀法证明核素能与人成骨细胞核仁中的Pp1δ结合。核仁组成的AgNORs和核素在对照的成骨细胞的细胞核中可见点状，在冈田酸处理的成骨细胞的细胞核中消失。在从成骨细胞获得的蛋白质中检测到一条110 kDa的主带。在凋亡的成骨细胞中，110 kDa蛋白的表达水平降低，而在冈田酸诱导的成骨细胞中，该蛋白的表达水平增加，并出现一条80 kDa的额外条带。本研究结果表明，成骨细胞核仁中的Pp1δ直接与核仁结合，核仁在成骨细胞的凋亡过程中发生裂解。

Protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell function. Okadaic acid is a potent inhibitor of PP1 and PP2A and induces apoptosis in human or mouse cells including osteoblasts. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in the nucleolus. The staining pattern of nucleolin in cultured osteoblastic cells is similar to that of PP1δ. Nucleolin was demonstrated to bind to PP1δ in nucleolus in human osteoblastic cells by using immunocytochemical and immunoprecipitation methods. AgNORs and nucleolin, visible as dots in the nuclei of the control osteoblastic cells, disappeared from the nuclei of the osteoblastic cells treated with okadaic acid. A major band, 110 kDa, was detected in the proteins obtained from osteoblastic cells. The level of the 110 kDa protein decreased in the apoptotic osteoblastic cells, whereas an additional band, 80 kDa, appeared and the level of this protein increased in the proteins prepared from okadaic acid-induced apoptotic osteoblastic cells. The present results indicate that PP1δ directly binds to nucleolin in the nucleolus of osteoblastic cells and that nucleolin is cleaved during apoptosis in osteoblastic cells.