Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity.
OBJECTIVE
Cdx2 is critical in intestinal proliferation and differentiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activation domain can modulate its function.


METHODS
The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fused to the Gal4-DNA binding domain. In vivo phosphorylation was studied by metabolic labeling with (32)P-orthophosphate. To study a potential phosphorylation site, polyclonal antibodies were generated: CNL was raised against amino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which serine 60 was phosphorylated.


RESULTS
A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine reduces incorporation of (32)P-orthophosphate substantially. S60-phosphorylation decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhibited with the mitogen-activated protein kinase inhibitors PD98059 or UO126. P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative compartment of the intestinal epithelial layer. In contrast, CNL recognizes Cdx2 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 have different transcriptional activity, as well as different spatial expression patterns in the intestinal epithelium.
Rings
E H
EH
Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Boudreau
F
F
Taylor
J K
JK
Moffett
J
J
Suh
E R
ER
Traber
P G
PG
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000071616:CDX2 Transcription Factor; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators; D015533:Transcriptional Activation / Q000502:physiology*
D000071616:CDX2 Transcription Factor; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D015534:Trans-Activators; D012694:Serine; D020928:Mitogen-Activated Protein Kinases