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

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.

FIGURE 1.

The c-Myc binding domain is necessary for transcriptional activation of cyclin B1 by cyclin G1. A, siRNA of scrambled control or cyclin G1 was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and Western blot analysis was performed. The relative band intensity was then calculated from densitometric scans of the immunoblots, with control values set to 1. The results represent one of three independent experiments. B, wild-type (WT) or various point or deletion (Δ) mutants of cyclin B1-Luc vectors were transfected to MFG control and cyclin G1-overexpressing cells, and a luciferase assay was then performed (top). Scrambled control (Si-Con) or siRNAs were transfected into cyclin G1-overexpressing cells (Si-Cyclin G1), and a cyclin B1 promoter assay was then performed using a luciferase assay (bottom). The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05.

FIGURE 2.

Involvement of c-Myc in cyclin G1-mediated transcriptional activation of cyclin B1. siRNAs of scrambled control (Si-Con), cyclin G1 (Si-Cyclin G1), cyclin B1 (Si-Cyclin B1), or c-Myc (Si-c-Myc) were transfected to MFG control and cyclin G1-overexpressing cells, and Western blotting (A) and luciferase assay (B) were then performed. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05 C, siRNA of scrambled control or c-Myc was transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells, and Western blot analysis was then performed (left). A gel mobility shift analysis was done using a wild-type E-box (WT-E-box) or mutant E-box (Mut-E-box) probe on the extracts of the MFG control or cyclin G1-transfected NCI-H460 cells with or without transfection siRNA of c-Myc. Nuclear extracts were prepared, and the c-Myc DNA binding activity was then measured using ³²P-labeled E-box fragments (right). Representative results from three independent experiments are shown.

FIGURE 3.

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.

FIGURE 4.

Increased phosphorylation of c-Myc on Ser-62 by cyclin G1. A, Western blot analysis of the phosphorylated forms of c-Myc (Ser-62 and Thr-58) in control and cyclin G1-overexpressing cells B, immunoblotting (IB) analysis of the phosphorylated forms of c-Myc (Ser-62 and Thr-58) following immunoprecipitation (IP: c-Myc) was performed in MFG control and cyclin G1-overexpressing cells C, ChIP-quantitative PCR analysis was performed to determine the in vivo binding of c-Myc to the cyclin B1 promoter. The α-globin promoter was used as a negative control in the ChIP-quantitative PCR (left). A ChIP assay was conducted in NCI-H460 cells or in cells transfected with various point mutant plasmids of c-Myc as reported under “Experimental Procedures.” Amplification was performed using oligonucleotides specific to the cyclin B1 (region –329/–3) gene. D, NCI-H460 cells were transiently transfected with control or mutant c-Myc plasmids. Western blot analysis and luciferase assays were performed, and the results are presented as the means ± S.D. of three independent experiments. E, protein extracts were prepared at the indicated time points following cycloheximide treatment (20 μg/ml) of NCI-H460 cells transfected with pcDNA3 and various point mutant plasmids of c-Myc that were hemagglutinin (HA)-tagged, and Western blot analysis was then performed using anti-hemagglutinin antibody. The relative band intensity was calculated from densitometric scans of immunoblots, with the control value set to 1. The results represent one of three independent experiments.

FIGURE 5.

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.

FIGURE 6.

Amino-terminal sequence of c-Myc is the binding site for interaction with Cdk5. A, pcDNA3 and c-Myc were transfected to H460 cells, and Western blot or immunoblotting (IB) was conducted following immunoprecipitation (IP). B, various deletion constructs of hemagglutinin (HA)-tagged c-Myc (left) were transfected to H460 cells. Western blot or immunoblotting was performed following immunoprecipitation (right).

FIGURE 7.

Inhibition of cyclin G1-mediated radiation sensitivity by knockdown of Cdk5. A, siRNAs from scrambled control (Si-Con) or Cdk5 (Si-Cdk5) were transfected to MFG control and cyclin G1-overexpressing NCI-H460 cells. Following overnight treatment with 200 nm nocodazole, cells were cultured with new media. Western blot analysis (top) and flow cytometry (bottom) were then performed at the indicated time points. B, Western blot or kinase assay of NCI-H460 cells transfected with siRNA of control, cyclin G1, or Cdk5 was conducted at the indicated time points following treatment with 5-Gy radiation. The results represent one of three independent experiments. *, denotes kinase assay. IP, immunoprecipitation. C, flow cytometric analysis after propidium iodide staining was conducted following 48 h of 5-Gy radiation using MFG control and cyclin G1-overexpressing NCI-H460 cells. The results represent the means ± S.D. of three independent experiments. *, denotes statistical significance of p < 0.05.

FIGURE 8.

Hypothetical scheme of Cdk5-mediated c-Myc phosphorylation on Ser-62 in cyclin G1-overexpressing cells. Cdk5, the binding partner of cyclin G1, was responsible for c-Myc phosphorylation on Ser-62, and this stable form of phosphorylated c-Myc then bound to the E-box promoter region of cyclin B1, which resulted in increased radiation-induced M phase arrest and cell death in cyclin G1-overexpressing cells.