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Cdk5-mediated phosphorylation of c-Myc on Ser-62 is essential in transcriptional activation of cyclin B1 by cyclin G1.

Seo HR, Kim J, Bae S, Soh JW, Lee YS - J. Biol. Chem. (2008)

Bottom Line: It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1.Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated.Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea.

ABSTRACT
It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

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Increased protein stability of c-Myc by cyclin G1. A, protein extracts were prepared at the indicated time points following cycloheximide treatment (20 μg/ml) of MFG control and cyclin G1-overexpressing NCI-H460 cells. Western blotting was performed using anti-c-Myc (top), and the relative band intensity was then calculated by comparing densitometric scans of the sample immunoblots with the values of control samples, which were set to 1 (bottom). The results represent one of three independent experiments. B, protein synthesis was conducted in MFG control and cyclin G1-overexpressing NCI-H460 cells with [35S]methionine for 30 min. Samples were collected every 10 min and analyzed to determine the rate of protein synthesis (top). Relative band intensity was calculated by comparing densitometric scans of the sample blots with those of control samples (0 time point), which were set to 1 (bottom). Means ± S.D. is shown.
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fig3: Increased protein stability of c-Myc by cyclin G1. A, protein extracts were prepared at the indicated time points following cycloheximide treatment (20 μg/ml) of MFG control and cyclin G1-overexpressing NCI-H460 cells. Western blotting was performed using anti-c-Myc (top), and the relative band intensity was then calculated by comparing densitometric scans of the sample immunoblots with the values of control samples, which were set to 1 (bottom). The results represent one of three independent experiments. B, protein synthesis was conducted in MFG control and cyclin G1-overexpressing NCI-H460 cells with [35S]methionine for 30 min. Samples were collected every 10 min and analyzed to determine the rate of protein synthesis (top). Relative band intensity was calculated by comparing densitometric scans of the sample blots with those of control samples (0 time point), which were set to 1 (bottom). Means ± S.D. is shown.

Mentions: Protein Synthesis—For protein synthesis assays, MFG control and cyclin G1-overexpressing NCI-H460 cells were resuspended in Met/Cys-free Dulbecco's modified Eagle's medium plus 2% dialyzed fetal bovine serum and then preincubated for 30 min at 37 °C for recovery. The cells were then labeled with [35S]methionine (5 Ci, 3000 Ci/mmol) for the times indicated in Fig. 3, after which 500 μl of cell suspension were lysed in SDS sample buffer and then incubated with c-Myc antibody. Next, the immunocomplexes were collected on protein A-Sepharose beads and then separated on SDS-polyacrylamide gels, after which the bands were detected by autoradiography.


Cdk5-mediated phosphorylation of c-Myc on Ser-62 is essential in transcriptional activation of cyclin B1 by cyclin G1.

Seo HR, Kim J, Bae S, Soh JW, Lee YS - J. Biol. Chem. (2008)

Increased protein stability of c-Myc by cyclin G1. A, protein extracts were prepared at the indicated time points following cycloheximide treatment (20 μg/ml) of MFG control and cyclin G1-overexpressing NCI-H460 cells. Western blotting was performed using anti-c-Myc (top), and the relative band intensity was then calculated by comparing densitometric scans of the sample immunoblots with the values of control samples, which were set to 1 (bottom). The results represent one of three independent experiments. B, protein synthesis was conducted in MFG control and cyclin G1-overexpressing NCI-H460 cells with [35S]methionine for 30 min. Samples were collected every 10 min and analyzed to determine the rate of protein synthesis (top). Relative band intensity was calculated by comparing densitometric scans of the sample blots with those of control samples (0 time point), which were set to 1 (bottom). Means ± S.D. is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2414302&req=5

fig3: Increased protein stability of c-Myc by cyclin G1. A, protein extracts were prepared at the indicated time points following cycloheximide treatment (20 μg/ml) of MFG control and cyclin G1-overexpressing NCI-H460 cells. Western blotting was performed using anti-c-Myc (top), and the relative band intensity was then calculated by comparing densitometric scans of the sample immunoblots with the values of control samples, which were set to 1 (bottom). The results represent one of three independent experiments. B, protein synthesis was conducted in MFG control and cyclin G1-overexpressing NCI-H460 cells with [35S]methionine for 30 min. Samples were collected every 10 min and analyzed to determine the rate of protein synthesis (top). Relative band intensity was calculated by comparing densitometric scans of the sample blots with those of control samples (0 time point), which were set to 1 (bottom). Means ± S.D. is shown.
Mentions: Protein Synthesis—For protein synthesis assays, MFG control and cyclin G1-overexpressing NCI-H460 cells were resuspended in Met/Cys-free Dulbecco's modified Eagle's medium plus 2% dialyzed fetal bovine serum and then preincubated for 30 min at 37 °C for recovery. The cells were then labeled with [35S]methionine (5 Ci, 3000 Ci/mmol) for the times indicated in Fig. 3, after which 500 μl of cell suspension were lysed in SDS sample buffer and then incubated with c-Myc antibody. Next, the immunocomplexes were collected on protein A-Sepharose beads and then separated on SDS-polyacrylamide gels, after which the bands were detected by autoradiography.

Bottom Line: It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1.Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated.Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea.

ABSTRACT
It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

Show MeSH