Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed‐2 leading to decreased DNA binding

Abstract
Engrailed‐2 (En‐2) belongs to an evolutionarily conserved family of DNA binding homeodomain‐containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En‐2 is phosphorylated by protein kinase A (PKA) in forskolin‐treated COS‐7 cells. Furthermore, we analyze the physiological function of En‐2 phosphorylation by PKA. The nuclear localization of En‐2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En‐2 to DNA, while replacing serine 267 with glutamic acid resulted in decreased En‐2 DNA binding. These results suggest that the transcriptional activity of En‐2 is regulated by PKA.