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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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Molecular models of S163 and S177. A molecular model of the PRH homeodomain–DNA complex based on a 3D alignment of the PRH homeodomain structure (PDB 1WQ1) and the homeodomain protein–DNA complexes (PDB 1ENH, 1HDP and 1IG7) was made using 3D-superimpose in Strap (45) and visualized using PyMOL (46). (A) The figure highlights the proximity of S163 to the DNA backbone (thick red line). (B) The hydrogen bond network that links S177 to the DNA backbone via Q180. In both images, the pink shading indicates the volume that would be occupied by phosphoserine.
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Figure 7: Molecular models of S163 and S177. A molecular model of the PRH homeodomain–DNA complex based on a 3D alignment of the PRH homeodomain structure (PDB 1WQ1) and the homeodomain protein–DNA complexes (PDB 1ENH, 1HDP and 1IG7) was made using 3D-superimpose in Strap (45) and visualized using PyMOL (46). (A) The figure highlights the proximity of S163 to the DNA backbone (thick red line). (B) The hydrogen bond network that links S177 to the DNA backbone via Q180. In both images, the pink shading indicates the volume that would be occupied by phosphoserine.

Mentions: Here, we have shown that human and avian PRH bind to the β subunit of CK2 and that PRH is phosphorylated by CK2 in vivo. Moreover, we have demonstrated that there is a decrease in PRH target gene expression in the presence of an inhibitor of CK2. This suggests that a functional consequence of phosphorylation of PRH by CK2 is the inhibition of transcriptional repression by PRH. We have shown that CK2 phosphorylates PRH at two sites within the homeodomain. Phosphorylation at these sites inhibits the DNA-binding activity of PRH in vitro and inhibits the transcription repression function of this protein in cells. Although several other homeodomain proteins are substrates for CK2, in these cases phosphorylation generally occurs outside the homeodomain. The cooperative binding to DNA of homeodomain proteins Antennapaedia and Extradenticle, for example, is regulated by CK2 phosphorylation of Antennaepaedia at multiple sites outside the homeodomain (5). Similarly, Engrailed is phosphorylated by CK2 at sites outside the homeodomain but in this case phosphorylation results in increased DNA-binding activity (8) and the inhibition of inter-cellular trafficking (7). In contrast, phosphorylation of Csx/Nk2.5 by CK2 occurs within the homeodomain at a consensus CK2 site ([S/T]XX[E/D]) conserved among all NK class homeodomain proteins and identified here as a phosphorylation site S163 in PRH (11). However, in the case of Csx/Nk2.5 phosphorylation results in increased DNA binding (11), S163 is located at the start of the second helix of the PRH homeodomain and this residue is not thought to be involved in binding to DNA. However, model building suggests that this amino acid in PRH could be in close approach to the phosphate backbone in the PRH–DNA complex (Figure 7A). Phosphorylation at this position in PRH would thus place a negative charge close to the DNA phosphate backbone and this would be expected to result in decreased DNA binding. Our mutagenesis data shows that the replacement of S163 with glutamic acid to mimic phosphorylation decreases the DNA-binding activity of PRH but is not sufficient to completely block DNA binding. Surface exposed glutamic acid residues can occupy several positions due to the flexibility of the side chain, and this might explain why DNA binding is reduced but not abolished by this substitution.Figure 7.


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)

Molecular models of S163 and S177. A molecular model of the PRH homeodomain–DNA complex based on a 3D alignment of the PRH homeodomain structure (PDB 1WQ1) and the homeodomain protein–DNA complexes (PDB 1ENH, 1HDP and 1IG7) was made using 3D-superimpose in Strap (45) and visualized using PyMOL (46). (A) The figure highlights the proximity of S163 to the DNA backbone (thick red line). (B) The hydrogen bond network that links S177 to the DNA backbone via Q180. In both images, the pink shading indicates the volume that would be occupied by phosphoserine.
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Figure 7: Molecular models of S163 and S177. A molecular model of the PRH homeodomain–DNA complex based on a 3D alignment of the PRH homeodomain structure (PDB 1WQ1) and the homeodomain protein–DNA complexes (PDB 1ENH, 1HDP and 1IG7) was made using 3D-superimpose in Strap (45) and visualized using PyMOL (46). (A) The figure highlights the proximity of S163 to the DNA backbone (thick red line). (B) The hydrogen bond network that links S177 to the DNA backbone via Q180. In both images, the pink shading indicates the volume that would be occupied by phosphoserine.
Mentions: Here, we have shown that human and avian PRH bind to the β subunit of CK2 and that PRH is phosphorylated by CK2 in vivo. Moreover, we have demonstrated that there is a decrease in PRH target gene expression in the presence of an inhibitor of CK2. This suggests that a functional consequence of phosphorylation of PRH by CK2 is the inhibition of transcriptional repression by PRH. We have shown that CK2 phosphorylates PRH at two sites within the homeodomain. Phosphorylation at these sites inhibits the DNA-binding activity of PRH in vitro and inhibits the transcription repression function of this protein in cells. Although several other homeodomain proteins are substrates for CK2, in these cases phosphorylation generally occurs outside the homeodomain. The cooperative binding to DNA of homeodomain proteins Antennapaedia and Extradenticle, for example, is regulated by CK2 phosphorylation of Antennaepaedia at multiple sites outside the homeodomain (5). Similarly, Engrailed is phosphorylated by CK2 at sites outside the homeodomain but in this case phosphorylation results in increased DNA-binding activity (8) and the inhibition of inter-cellular trafficking (7). In contrast, phosphorylation of Csx/Nk2.5 by CK2 occurs within the homeodomain at a consensus CK2 site ([S/T]XX[E/D]) conserved among all NK class homeodomain proteins and identified here as a phosphorylation site S163 in PRH (11). However, in the case of Csx/Nk2.5 phosphorylation results in increased DNA binding (11), S163 is located at the start of the second helix of the PRH homeodomain and this residue is not thought to be involved in binding to DNA. However, model building suggests that this amino acid in PRH could be in close approach to the phosphate backbone in the PRH–DNA complex (Figure 7A). Phosphorylation at this position in PRH would thus place a negative charge close to the DNA phosphate backbone and this would be expected to result in decreased DNA binding. Our mutagenesis data shows that the replacement of S163 with glutamic acid to mimic phosphorylation decreases the DNA-binding activity of PRH but is not sufficient to completely block DNA binding. Surface exposed glutamic acid residues can occupy several positions due to the flexibility of the side chain, and this might explain why DNA binding is reduced but not abolished by this substitution.Figure 7.

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.

Show MeSH