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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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Cdk5 activation by cyclin G1 phosphorylates c-Myc on Ser-62. A, siRNA of scrambled control (Si-Cont), cyclin G1 (Si-Cyclin G1), cyclin B1 (Si-Cyclin B1), or Cdk5 (Si-Cdk5) were transfected to MFG control and cyclin G1-overexpressing cells. Western blot (WB), immunoblotting, or kinase assays were performed following immunoprecipitation (IP). *, denotes kinase assay; **, denotes immunoprecipitation assay. B, siRNA of scrambled control or Cdk5 was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and a cyclin B1 promoter assay was performed by luciferase assay. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05 C, an in vitro kinase assay was conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. D, an in vitro translation and a GST pulldown assay were conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. E, Western blotting was performed using NCI-H596 cell lysates with or without GST-cyclin G1 or GST-Cdk5 recombinant proteins.
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fig5: Cdk5 activation by cyclin G1 phosphorylates c-Myc on Ser-62. A, siRNA of scrambled control (Si-Cont), cyclin G1 (Si-Cyclin G1), cyclin B1 (Si-Cyclin B1), or Cdk5 (Si-Cdk5) were transfected to MFG control and cyclin G1-overexpressing cells. Western blot (WB), immunoblotting, or kinase assays were performed following immunoprecipitation (IP). *, denotes kinase assay; **, denotes immunoprecipitation assay. B, siRNA of scrambled control or Cdk5 was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and a cyclin B1 promoter assay was performed by luciferase assay. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05 C, an in vitro kinase assay was conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. D, an in vitro translation and a GST pulldown assay were conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. E, Western blotting was performed using NCI-H596 cell lysates with or without GST-cyclin G1 or GST-Cdk5 recombinant proteins.

Mentions: Activation of Cdk5 by Cyclin G1 Phosphorylates c-Myc on Ser-62—The activity of Cdk5, a binding partner of cyclin G1, was increased in cyclin G1-overexpressing cells (supplemental Fig. 3). In addition, treatment of the cells with Si-Cdk5 or Sicyclin G1 inhibited this kinase activity when c-Myc protein was used as a substrate. Increased phosphorylation of c-Myc on Ser-62 and cyclin B1 protein expression in cyclin G1-overexpressing cells were also inhibited by treatment with Si-Cdk5 or Si-cyclin G1. This suggests that Cdk5 is involved in cyclin G1-mediated c-Myc phosphorylation and cyclin B1 expression, as well as in cyclin B1 transcriptional activity (Fig. 5, A and B). To determine whether cyclin G1 or Cdk5 directly regulated c-Myc phosphorylation, an in vitro kinase assay was performed. Treatment with GST-cyclin G1 protein alone did not induce 32P incorporation when c-Myc was used as a substrate. However, treatment with GST-Cdk5 protein induced c-Myc phosphorylation, and co-treatment with GST-cyclin G1 and GST-Cdk5 protein enhanced 32P incorporation, which suggests that Cdk5 is responsible for c-Myc phosphorylation and co-treatment of cyclin G1 and that Cdk5 potentiates this phenomenon (Fig. 5C). An in vitro translational assay using a TnT kit also indicated that Cdk5 directly bound to c-Myc and that cyclin G1 potentiated this binding (Fig. 5D). Furthermore, addition of GST-cyclin G1 protein to the cell lysates of NCI-H596 resulted in a lower expression of cyclin G1, which in turn increased the level of c-Myc phosphorylation on Ser-62 via activation of Cdk5 by cyclin G1. Additionally, cell lysates that were treated with GST-Cdk5 protein showed greater c-Myc phosphorylation than those treated with only GST-cyclin G1 protein, and co-treatment with GST-cyclin G1 and GST-Cdk5 proteins dramatically increased these phenomena (Fig. 5E). Taken together, these results indicate that cyclin G1-mediated c-Myc phosphorylation on Ser-62 and cyclin B1 expression resulted from Cdk5 activation by cyclin G1.


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)

Cdk5 activation by cyclin G1 phosphorylates c-Myc on Ser-62. A, siRNA of scrambled control (Si-Cont), cyclin G1 (Si-Cyclin G1), cyclin B1 (Si-Cyclin B1), or Cdk5 (Si-Cdk5) were transfected to MFG control and cyclin G1-overexpressing cells. Western blot (WB), immunoblotting, or kinase assays were performed following immunoprecipitation (IP). *, denotes kinase assay; **, denotes immunoprecipitation assay. B, siRNA of scrambled control or Cdk5 was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and a cyclin B1 promoter assay was performed by luciferase assay. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05 C, an in vitro kinase assay was conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. D, an in vitro translation and a GST pulldown assay were conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. E, Western blotting was performed using NCI-H596 cell lysates with or without GST-cyclin G1 or GST-Cdk5 recombinant proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Cdk5 activation by cyclin G1 phosphorylates c-Myc on Ser-62. A, siRNA of scrambled control (Si-Cont), cyclin G1 (Si-Cyclin G1), cyclin B1 (Si-Cyclin B1), or Cdk5 (Si-Cdk5) were transfected to MFG control and cyclin G1-overexpressing cells. Western blot (WB), immunoblotting, or kinase assays were performed following immunoprecipitation (IP). *, denotes kinase assay; **, denotes immunoprecipitation assay. B, siRNA of scrambled control or Cdk5 was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and a cyclin B1 promoter assay was performed by luciferase assay. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05 C, an in vitro kinase assay was conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. D, an in vitro translation and a GST pulldown assay were conducted using cyclin G1, Cdk5, and c-Myc recombinant proteins. E, Western blotting was performed using NCI-H596 cell lysates with or without GST-cyclin G1 or GST-Cdk5 recombinant proteins.
Mentions: Activation of Cdk5 by Cyclin G1 Phosphorylates c-Myc on Ser-62—The activity of Cdk5, a binding partner of cyclin G1, was increased in cyclin G1-overexpressing cells (supplemental Fig. 3). In addition, treatment of the cells with Si-Cdk5 or Sicyclin G1 inhibited this kinase activity when c-Myc protein was used as a substrate. Increased phosphorylation of c-Myc on Ser-62 and cyclin B1 protein expression in cyclin G1-overexpressing cells were also inhibited by treatment with Si-Cdk5 or Si-cyclin G1. This suggests that Cdk5 is involved in cyclin G1-mediated c-Myc phosphorylation and cyclin B1 expression, as well as in cyclin B1 transcriptional activity (Fig. 5, A and B). To determine whether cyclin G1 or Cdk5 directly regulated c-Myc phosphorylation, an in vitro kinase assay was performed. Treatment with GST-cyclin G1 protein alone did not induce 32P incorporation when c-Myc was used as a substrate. However, treatment with GST-Cdk5 protein induced c-Myc phosphorylation, and co-treatment with GST-cyclin G1 and GST-Cdk5 protein enhanced 32P incorporation, which suggests that Cdk5 is responsible for c-Myc phosphorylation and co-treatment of cyclin G1 and that Cdk5 potentiates this phenomenon (Fig. 5C). An in vitro translational assay using a TnT kit also indicated that Cdk5 directly bound to c-Myc and that cyclin G1 potentiated this binding (Fig. 5D). Furthermore, addition of GST-cyclin G1 protein to the cell lysates of NCI-H596 resulted in a lower expression of cyclin G1, which in turn increased the level of c-Myc phosphorylation on Ser-62 via activation of Cdk5 by cyclin G1. Additionally, cell lysates that were treated with GST-Cdk5 protein showed greater c-Myc phosphorylation than those treated with only GST-cyclin G1 protein, and co-treatment with GST-cyclin G1 and GST-Cdk5 proteins dramatically increased these phenomena (Fig. 5E). Taken together, these results indicate that cyclin G1-mediated c-Myc phosphorylation on Ser-62 and cyclin B1 expression resulted from Cdk5 activation by cyclin G1.

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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