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 language | English |
|---|---|
| Pages (from-to) | 369-375 |
| Number of pages | 7 |
| Journal | Cell Biochemistry and Function |
| Volume | 25 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Jul 2007 |
| Externally published | Yes |
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Keywords
- 2D-IEF-PAGE
- Cell cycle
- MG63 cell
- Nucleolin
- Protein phosphatases
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology
Cite this
Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest. / Morimoto, Hiroyuki; Ozaki, Akiko; Okamura, Hirohiko; Yoshida, Kaya; Amorim, Bruna Rabelo; Tanaka, Hiroaki; Kitamura, Seiichiro; Haneji, Tatsuji.
In: Cell Biochemistry and Function, Vol. 25, No. 4, 07.2007, p. 369-375.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest
AU - Morimoto, Hiroyuki
AU - Ozaki, Akiko
AU - Okamura, Hirohiko
AU - Yoshida, Kaya
AU - Amorim, Bruna Rabelo
AU - Tanaka, Hiroaki
AU - Kitamura, Seiichiro
AU - Haneji, Tatsuji
PY - 2007/7
Y1 - 2007/7
N2 - 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.
AB - 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.
KW - 2D-IEF-PAGE
KW - Cell cycle
KW - MG63 cell
KW - Nucleolin
KW - Protein phosphatases
UR - http://www.scopus.com/inward/record.url?scp=34447521586&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34447521586&partnerID=8YFLogxK
U2 - 10.1002/cbf.1300
DO - 10.1002/cbf.1300
M3 - Article
C2 - 16329155
AN - SCOPUS:34447521586
VL - 25
SP - 369
EP - 375
JO - Cell Biochemistry and Function
JF - Cell Biochemistry and Function
SN - 0263-6484
IS - 4
ER -