Protein dephosphorylation and apoptosis in osteoblasts

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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.