Studies on the homeodomain transcription factor Phox2a: Regulation of the phosphorylation status of Phox2a by the cAMP pathway

Homeodomain transcription factor Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27Kip1 transcription in response to cAMP signaling. To demonstrate the mechanism of Phox2a activation by cAMP signailng, Phox2a phosphorylation sites were identified by mass spectrometry. Ser206 was identified as the most abundant phosphorylation site in Phox2a. A phospho-Ser206 Phox2a antibody detected transient dephosphorylation of Phox2a upon neuronal differentiation of noradrenergic CAD cells, dependent on the activation of the cAMP pathway.;Employing serine to alanine and serine to aspartic acid Phox2a mutants expressed in tetracycline-regulated CAD cell lines, Phox2a transcriptional activity is regulated by two cAMP-dependent events: (1) cAMP signaling promotes the dephosphorylation of Phox2a Ser206, thereby allowing Phox2a to bind p27 Kip1 promoter and promotes p27Kip1 transcription. (2) Phox2a becomes phosphorylated by PKA on Ser153 after Ser202 and Ser208 are dephosphorylated. This prevents the association with p27Kip1 promoter and terminates p27Kip1 transcription. Taken together, this study demonstrates a novel time-dependent Phox2a transcriptional mechanism by the cAMP signaling pathway.