Differential effects of phosphorylation on DNA binding properties of N Oct-3 are dictated by protein/DNA complex structures.
N Oct-3, a transcription factor member of the POU protein family, is implicated in normal central nervous system development but also in melanoma growth. Its DNA-binding domain (DBD) comprises two subdomains, POUs and POUh, joined by a linker peptide. We have previously shown that N Oct-3 can interact with the already described PORE and MORE DNA motifs, but also with a new structural element we have termed NORE. Having observed that both the PORE and NORE DNA-association modes depend on a strong anchoring of the POUh subdomain rigid arm into the DNA-target minor groove, in contrast to the MORE mode, we have formulated the hypothesis that phosphorylation of the conserved Ser101 residue located in the N Oct-3 POUh arm could lead to differential results in DNA binding according to the type of target. Here we demonstrate that, in vitro, Ser101 is phosphorylated by protein kinase A (PKA), either purified or contained in melanoma (624 mel) nuclear extract, and that this phosphorylation indeed significantly reduced N Oct-3 DBD binding to PORE and NORE motifs, most likely by hampering the POUh rigid arm insertion in the DNA minor groove. Conversely, no effect was observed on the binding of N Oct-3 DBD to MORE sequences. Finally, once bound to its DNA targets, N Oct-3 DBD is less susceptible to PKA activity. We conclude that transcription of genes exhibiting a MORE motif in their promoter should be less affected by N Oct-3 phosphorylation than that of genes switched on by PORE or NORE sequences.
Nieto
Laurence
L
Interactions Acides Nucléiques, Protéines Comme Cibles Pharmacologiques, Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS/Université Paul Sabatier, Toulouse, France. laurence.nieto@ipbs.fr <laurence.nieto@ipbs.fr>
Joseph
Gérard
G
Stella
Alexandre
A
Henri
Pauline
P
Burlet-Schiltz
Odile
O
Monsarrat
Bernard
B
Clottes
Eric
E
Erard
Monique
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002457:Cell Extracts; D002467:Cell Nucleus / Q000201:enzymology; D003198:Computer Simulation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D050814:Octamer Transcription Factor-3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050809:POU Domain Factors / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002457:Cell Extracts; D050814:Octamer Transcription Factor-3; D050809:POU Domain Factors; D010768:Phosphoserine; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases