Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

Hiroyuki Morimoto, Akiko Ozaki, Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Hiroaki Tanaka, Seiichiro Kitamura, Tatsuji Haneji

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Abstract

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G1/S and G2/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G2/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G1/S and G2/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.

Original languageEnglish
Pages (from-to)369-375
Number of pages7
JournalCell biochemistry and function
Volume25
Issue number4
DOIs
Publication statusPublished - Jul 1 2007
Externally publishedYes

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Keywords

  • 2D-IEF-PAGE
  • Cell cycle
  • MG63 cell
  • Nucleolin
  • Protein phosphatases

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Cite this

Morimoto, H., Ozaki, A., Okamura, H., Yoshida, K., Amorim, B. R., Tanaka, H., Kitamura, S., & Haneji, T. (2007). Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest. Cell biochemistry and function, 25(4), 369-375. https://doi.org/10.1002/cbf.1300