The paired-like homeodomain protein, Arix, mediates protein kinase A-stimulated dopamine beta-hydroxylase gene transcription through its phosphorylation status.

The homeodomain transcription factor Arix/Phox2a plays a critical role in the specification of noradrenergic neurons by inducing the expression of dopamine beta-hydroxylase (DBH), the terminal enzyme for noradrenaline biosynthesis. In reporter assays, Arix together with activation of cAMP-dependent protein kinase (PKA) potentiates DBH gene transcription. We have evaluated whether post-translational modification of Arix regulates PKA-mediated DBH gene transcription. We found that Arix is constitutively phosphorylated in vivo at the basal level and that the phosphorylation level is substantially decreased upon stimulation of the PKA pathway. The change in the Arix phosphorylation state coincides with DNA binding activity of Arix. Treatment of cells with forskolin results in a robust enhancement of the DNA binding of Arix, which is reversed by treatment with serine/threonine and tyrosine phosphatase inhibitors. Consistent with the DNA binding activity of Arix, treatment of cultured cells with phosphatase inhibitors diminishes transcriptional activation with Arix plus forskolin. Amino acid analysis demonstrates the presence of phosphoserine within Arix. The results collectively suggest that dephosphorylation of Arix is a necessary event to fully activate PKA-mediated DBH transcription. Thus, the present study demonstrates that Arix can integrate extrinsic signals through post-translational modification, regulating DBH gene transcription in response to activation of the PKA pathway.