Differential effects of phosphorylation on DNA binding properties of N Oct-3 are dictated by protein/DNA complex...

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.