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CK2 phosphorylation of the PRH/Hex homeodomain functions as a reversible switch for DNA binding.

Soufi A, Noy P, Buckle M, Sawasdichai A, Gaston K, Jayaraman PS - Nucleic Acids Res. (2009)

Bottom Line: We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177.Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells.Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Research, Birmingham University Medical School, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the beta subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

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Phosphorylation of the homeodomain inhibits DNA binding. (A) A schematic representation of the full-length PRH protein (top line) and the truncated PRH proteins (second line) used in this study. Labels and numbering are as shown in Figure 1. The filled rectangle represents a histidine tag. The experiment shown in Figure 3 was repeated using the PRH-HDC (B) and PRH-HD (C) proteins.
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Figure 4: Phosphorylation of the homeodomain inhibits DNA binding. (A) A schematic representation of the full-length PRH protein (top line) and the truncated PRH proteins (second line) used in this study. Labels and numbering are as shown in Figure 1. The filled rectangle represents a histidine tag. The experiment shown in Figure 3 was repeated using the PRH-HDC (B) and PRH-HD (C) proteins.

Mentions: Inspection of the amino acid sequence of PRH reveals several consensus CK2 sites that are conserved between the human and avian proteins. To investigate the importance of these or other phosphorylation sites in the inhibition of DNA binding, we made use of two truncated PRH proteins consisting of the PRH homeodomain and C-terminal domain (PRH-HDC) and the PRH homeodomain alone (PRH-HD), respectively (Figure 4A). The effects of phosphorylation on the DNA-binding activity of these proteins were analysed using EMSA as described previously for the full-length protein. Figure 4B and C show that both PRH-HDC and PRH-HD are able to bind DNA. The DNA-binding activity of both of these proteins is somewhat reduced after incubation with CK2 alone. However, the DNA-binding activity of both proteins is abolished after incubation with both CK2 and ATP (Figure 4B and C, lane 4). Furthermore, in both cases incubation with CAP after treatment with CK2 and ATP, results in the restoration of DNA-binding activity (Figure 4B and C, lane 5). These data demonstrate that phosphorylation of the homeodomain alone is sufficient to inhibit the DNA-binding activity of PRH and that as in the case of the full-length protein, the effects of phosphorylation are readily reversible.Figure 4.


CK2 phosphorylation of the PRH/Hex homeodomain functions as a reversible switch for DNA binding.

Soufi A, Noy P, Buckle M, Sawasdichai A, Gaston K, Jayaraman PS - Nucleic Acids Res. (2009)

Phosphorylation of the homeodomain inhibits DNA binding. (A) A schematic representation of the full-length PRH protein (top line) and the truncated PRH proteins (second line) used in this study. Labels and numbering are as shown in Figure 1. The filled rectangle represents a histidine tag. The experiment shown in Figure 3 was repeated using the PRH-HDC (B) and PRH-HD (C) proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691835&req=5

Figure 4: Phosphorylation of the homeodomain inhibits DNA binding. (A) A schematic representation of the full-length PRH protein (top line) and the truncated PRH proteins (second line) used in this study. Labels and numbering are as shown in Figure 1. The filled rectangle represents a histidine tag. The experiment shown in Figure 3 was repeated using the PRH-HDC (B) and PRH-HD (C) proteins.
Mentions: Inspection of the amino acid sequence of PRH reveals several consensus CK2 sites that are conserved between the human and avian proteins. To investigate the importance of these or other phosphorylation sites in the inhibition of DNA binding, we made use of two truncated PRH proteins consisting of the PRH homeodomain and C-terminal domain (PRH-HDC) and the PRH homeodomain alone (PRH-HD), respectively (Figure 4A). The effects of phosphorylation on the DNA-binding activity of these proteins were analysed using EMSA as described previously for the full-length protein. Figure 4B and C show that both PRH-HDC and PRH-HD are able to bind DNA. The DNA-binding activity of both of these proteins is somewhat reduced after incubation with CK2 alone. However, the DNA-binding activity of both proteins is abolished after incubation with both CK2 and ATP (Figure 4B and C, lane 4). Furthermore, in both cases incubation with CAP after treatment with CK2 and ATP, results in the restoration of DNA-binding activity (Figure 4B and C, lane 5). These data demonstrate that phosphorylation of the homeodomain alone is sufficient to inhibit the DNA-binding activity of PRH and that as in the case of the full-length protein, the effects of phosphorylation are readily reversible.Figure 4.

Bottom Line: We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177.Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells.Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Research, Birmingham University Medical School, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the beta subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

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