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Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

Sakamoto K, Huang BW, Iwasaki K, Hailemariam K, Ninomiya-Tsuji J, Tsuji Y - Mol. Biol. Cell (2010)

Bottom Line: Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression.HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression.These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

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Replacement of CREB Serine 271 with glutamic acid caused the retarded migration in SDS-PAGE and enhanced CREB transactivation function. (A) CREB-WT, 133E, 271E mutant alone or CREB-WT plus Flag-HIPK2 was transfected into K562 cells. Whole cell lysates were prepared after 48 h and subjected to SDS-PAGE and Western blotting with anti-CREB antibody. Intact CREB proteins (retarded and nonretarded) are indicated with arrowheads. The smaller CREB proteins appear to be produced by unknown proteolytic cleavage. GAPDH Western blotting is shown as a loading control. (B) K562 cells were transfected with CREB-WT, CREB 133E, CREB 271E, or CREB-WT plus HIPK2. Whole cell lysates were treated with protein phosphatase (PPase+) before SDS-PAGE and Western blotting with anti-CREB antibody. (C) 0.1 or 0.3 μg of CREB-WT, Serine-to-glutamic acid mutant 133E or 271E expression plasmid was transfected with CRE4-luciferase reporter into K562 cells. Cells were harvested after 24 h incubation, and luciferase expression was measured. Luciferase expression from the reporter alone was set to 1.0. Average from eight independent experiments are shown with SEs.
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Figure 4: Replacement of CREB Serine 271 with glutamic acid caused the retarded migration in SDS-PAGE and enhanced CREB transactivation function. (A) CREB-WT, 133E, 271E mutant alone or CREB-WT plus Flag-HIPK2 was transfected into K562 cells. Whole cell lysates were prepared after 48 h and subjected to SDS-PAGE and Western blotting with anti-CREB antibody. Intact CREB proteins (retarded and nonretarded) are indicated with arrowheads. The smaller CREB proteins appear to be produced by unknown proteolytic cleavage. GAPDH Western blotting is shown as a loading control. (B) K562 cells were transfected with CREB-WT, CREB 133E, CREB 271E, or CREB-WT plus HIPK2. Whole cell lysates were treated with protein phosphatase (PPase+) before SDS-PAGE and Western blotting with anti-CREB antibody. (C) 0.1 or 0.3 μg of CREB-WT, Serine-to-glutamic acid mutant 133E or 271E expression plasmid was transfected with CRE4-luciferase reporter into K562 cells. Cells were harvested after 24 h incubation, and luciferase expression was measured. Luciferase expression from the reporter alone was set to 1.0. Average from eight independent experiments are shown with SEs.

Mentions: To further elucidate the role of HIPK2 in CREB phosphorylation at Serine 271, we generated the CREB Serine 271-to-glutamic acid mutant (271E) to mimic Serine 271 phosphorylated CREB. As a control, we generated the Serine 133 to glutamic acid mutant CREB (133E). First, we expressed these CREB mutants in K562 cells and tested whether the replacement of Serine with glutamic acid causes the retardation in SDS-PAGE. As shown in Figure 4A, 271E CREB showed the retarded migration that was similar to one induced by coexpression of wtCREB and HIPK2, whereas the 133E CREB mutant did not show the retarded migration. The retardation of 271E CREB was resistant to phosphatase treatment (Figure 4B), excluding the possibility of other CREB phosphorylation sites for the retardation. Then, we tested the effect of these glutamic acid CREB mutants on CRE-dependent transcription by a luciferase reporter assay. Compared with the CRE-dependent transcription activated by wtCREB or 133E CREB, 271E CREB activated it 1.8–2.0-fold higher than wt or 133E CREB (Figure 4C). No difference in the CREB activity between wt and 133E in this assay appears to be consistent with a previous report showing no significant impact of Serine 133 to glutamic acid replacement on CREB and coactivator interaction (Solt et al., 2006). Furthermore, cotransfection of wtCREB or Serine 271-to-Alanine mutant CREB (271A) along with HIPK2 in CRE4-luciferase assays demonstrated that the mutation of Serine 271 to Alanine diminished HIPK2-mediated activation (Figure 5A). Collectively, these results suggest that the phosphorylation of CREB at Serine 271 by HIPK2 enhanced the transactivation function of CREB.


Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

Sakamoto K, Huang BW, Iwasaki K, Hailemariam K, Ninomiya-Tsuji J, Tsuji Y - Mol. Biol. Cell (2010)

Replacement of CREB Serine 271 with glutamic acid caused the retarded migration in SDS-PAGE and enhanced CREB transactivation function. (A) CREB-WT, 133E, 271E mutant alone or CREB-WT plus Flag-HIPK2 was transfected into K562 cells. Whole cell lysates were prepared after 48 h and subjected to SDS-PAGE and Western blotting with anti-CREB antibody. Intact CREB proteins (retarded and nonretarded) are indicated with arrowheads. The smaller CREB proteins appear to be produced by unknown proteolytic cleavage. GAPDH Western blotting is shown as a loading control. (B) K562 cells were transfected with CREB-WT, CREB 133E, CREB 271E, or CREB-WT plus HIPK2. Whole cell lysates were treated with protein phosphatase (PPase+) before SDS-PAGE and Western blotting with anti-CREB antibody. (C) 0.1 or 0.3 μg of CREB-WT, Serine-to-glutamic acid mutant 133E or 271E expression plasmid was transfected with CRE4-luciferase reporter into K562 cells. Cells were harvested after 24 h incubation, and luciferase expression was measured. Luciferase expression from the reporter alone was set to 1.0. Average from eight independent experiments are shown with SEs.
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Figure 4: Replacement of CREB Serine 271 with glutamic acid caused the retarded migration in SDS-PAGE and enhanced CREB transactivation function. (A) CREB-WT, 133E, 271E mutant alone or CREB-WT plus Flag-HIPK2 was transfected into K562 cells. Whole cell lysates were prepared after 48 h and subjected to SDS-PAGE and Western blotting with anti-CREB antibody. Intact CREB proteins (retarded and nonretarded) are indicated with arrowheads. The smaller CREB proteins appear to be produced by unknown proteolytic cleavage. GAPDH Western blotting is shown as a loading control. (B) K562 cells were transfected with CREB-WT, CREB 133E, CREB 271E, or CREB-WT plus HIPK2. Whole cell lysates were treated with protein phosphatase (PPase+) before SDS-PAGE and Western blotting with anti-CREB antibody. (C) 0.1 or 0.3 μg of CREB-WT, Serine-to-glutamic acid mutant 133E or 271E expression plasmid was transfected with CRE4-luciferase reporter into K562 cells. Cells were harvested after 24 h incubation, and luciferase expression was measured. Luciferase expression from the reporter alone was set to 1.0. Average from eight independent experiments are shown with SEs.
Mentions: To further elucidate the role of HIPK2 in CREB phosphorylation at Serine 271, we generated the CREB Serine 271-to-glutamic acid mutant (271E) to mimic Serine 271 phosphorylated CREB. As a control, we generated the Serine 133 to glutamic acid mutant CREB (133E). First, we expressed these CREB mutants in K562 cells and tested whether the replacement of Serine with glutamic acid causes the retardation in SDS-PAGE. As shown in Figure 4A, 271E CREB showed the retarded migration that was similar to one induced by coexpression of wtCREB and HIPK2, whereas the 133E CREB mutant did not show the retarded migration. The retardation of 271E CREB was resistant to phosphatase treatment (Figure 4B), excluding the possibility of other CREB phosphorylation sites for the retardation. Then, we tested the effect of these glutamic acid CREB mutants on CRE-dependent transcription by a luciferase reporter assay. Compared with the CRE-dependent transcription activated by wtCREB or 133E CREB, 271E CREB activated it 1.8–2.0-fold higher than wt or 133E CREB (Figure 4C). No difference in the CREB activity between wt and 133E in this assay appears to be consistent with a previous report showing no significant impact of Serine 133 to glutamic acid replacement on CREB and coactivator interaction (Solt et al., 2006). Furthermore, cotransfection of wtCREB or Serine 271-to-Alanine mutant CREB (271A) along with HIPK2 in CRE4-luciferase assays demonstrated that the mutation of Serine 271 to Alanine diminished HIPK2-mediated activation (Figure 5A). Collectively, these results suggest that the phosphorylation of CREB at Serine 271 by HIPK2 enhanced the transactivation function of CREB.

Bottom Line: Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression.HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression.These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

Show MeSH
Related in: MedlinePlus