骨芽細胞と蛋白質脱リン酸化酵素 1 型 : 核小体での r-RNA 合成に関して

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Protein phosphorylation and dephosphorylation have been recognized as an essential mechanism in the regulation of cellular metabolism and functions in various tissues. cDNA cloning revealed the existence of at least four isoforms of PP1 catalytic subunit, termed PP1 α, PP1γ1, PP1γ2, and PP1δ. PP1 targeting subunit is thought to localize to specific subcellular component and to modulate the activity of the enzyme at these sites. In the present study, we examined the cytolocalization of PP1 isozymes and expression of these proteins in MG63 cells by immunofluorescent technique and Western blot analysis, respectively. PP1α and PP1δ antibodies recognized 38 kDa and 37 kDa proteins, respectively, present in the nuclear and nucleolar regions of MG63 cells, whereas PP1γ1 antibody interacted with 36 kDa protein in cytosolic fraction. This differential distribution of PP1s suggests that these isozymes may act in a sequential fashion on modulating the activity of this enzyme in MG63 cells. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in nucleolus. Nucleolin is directly involved in the regulation of ribosome biogenesis and maturation. Staining pattern of nucleolin in MG63 cells is similar to that of the PP1δ. PP1δ and nucleolin were specifically stained as dots in the nucleus. The multiple fluorescence image revealed that PP1δ and nucleolin represent same localization in nucleolus. To examine the interaction of PP1δ and nucleolin, whole cell lysates were immunoprecipitated with PP1δ antibody. The immunoprecipitant was analyzed by Western blotting using anti-nucleolin antibody. The anti-nucleolin antibody interacted with the protein which molecular weight was estimated as 100 kDa indicating. However, the sample precipitated with the normal rabbit serum was not stained with anti-nucleolin antibody. These results indicate that PP1δ associate with nucleolin directly at cellular nucleolus. MG 63 cells were treated with actinomycin D to inhibit r-RNA synthesis. In the actinomycin D-treated cells, subcellular localization of PP1δ and nucleolin were changed. The amount of PP1δ increased whereas the level of and dephosphorylated nucleolin increased. MG63 cells were also blocked in G1/S and G2/M phase by exposing them to hydroxyurea and nocodazole, respectively, and followed by immunoblotting. The intensity of immunoreaction of PP1α, PP1δ and PP1γ1 were different between the proteins prepared from the cells at G1/S and G2/M phase. PP1α increased at G1/S and G2/M phase, PP1δ increased at G1/S and G2/M phase with the intensity was higher at G2/M phase. However, the level of PP1γ1 increased only at G1/M phase. The present results indicate that nucleolin is one of the candidate of PP1δ substrate. The present results also indicate that PP1 is involved in r-RNA synthesis and in cell cycle regulation.
九州歯科学会の論文
2000-10-25

骨芽細胞と蛋白質脱リン酸化酵素 1 型 : 核小体での r-RNA 合成に関して

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