Phosphorylation of the serine 60 residue within the Cdx2 activation domainmediates its transactivation capacity

Background & Aims: Cdx2 is critical in intestinal proliferation and differe ntiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activatio n domain can modulate its function. Methods: The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fus ed to the Gal4-DNA binding domain. In vivo phosphorylation was studied by m etabolic labeling with P-32-orthophosphate. To study a potential phosphoryl ation site, polyclonal antibodies were generated: CNL was raised against am ino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which se rine 60 was phosphorylated. Results: A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine re duces incorporation of P-32-orthophosphate substantially. S60-phosphorylati on decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhi bited with the mitogen-activated protein kinase inhibitors PD98059 or U0126 . P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative co mpartment of the intestinal epithelial layer. In contrast, CNL recognizes C dx2 predominantly in the differentiated compartment. Conclusions: The Cdx2 activation domain is phosphorylated at serine 60 via the mitogen-activated protein kinase pathway. S60-phosphorylated and S60-nonphosphorylated Cdx2 h ave different transcriptional activity, as well as different spatial expres sion patterns in the intestinal epithelium.