Volume 121, Issue 6, Pages 1437-1450 (December 2001) Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity  Edmond H.H.M. Rings, François Boudreau, Jennifer K. Taylor, Jennifer Moffett, Eun Ran Suh, Peter G. Traber  Gastroenterology  Volume 121, Issue 6, Pages 1437-1450 (December 2001) DOI: 10.1053/gast.2001.29618 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Mapping of the Cdx2 transactivation domain. (A) NIH-3T3 cells were transfected with 1 μg of the 10xGal4-TK-luc reporter vector, as well as the different Gal4-Cdx2 expression plasmids and CMV-βgal for normalization of transfection. The Gal4-DBD, the A, B, and C boxes that are conserved regions in caudal homeobox proteins, and the homeodomain (HD) are indicated. The amounts of Gal4-DBD-Cdx2 expression plasmids used were adjusted to normalize the quantity of Gal4-DBD-Cdx2 fusion protein expression determined by immunoblots to the Gal4-DBD (data not shown). Relative luciferase activity is shown with Gal4-Cdx2 (50–140) set at 100%. In addition, transcriptional activity of a deletion protein lacking amino acid residues 54–137 was also compared with the full-length Cdx2 protein, using the sucrase-isomaltase promoter linked to a luciferase reporter. The full-length Cdx2 protein was set at 100%. (B) The amounts of pRC/CMV Cdx2 expression plasmids used were adjusted to normalize the quantity of Cdx2 protein expression determined by immunoblots. (C) Binding of Cdx2 and Cdx2 (Δ54-137) to the Cdx2-specific SIF1 DNA sequence. Nuclear extracts from NIH-3T3 cells transfected with Cdx2 (Δ54-137) (lanes 2–3) or Cdx2 (lanes 4–5) were incubated with labeled SIF1 DNA (lane 1, probe alone). Complex A (Cdx2 monomers) and B (Cdx2 dimers) were formed. Size differences between Cdx2 and Cdx2 (Δ54-137) regarding the formation of complex A and B are indicated. Complexes were supershifted (arrow) with the Cdx2 Cterm (*) antibody. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Further analysis of the Cdx2 activation domain. (A) Diagram of the functional domains of Cdx2. The activation domain, the A, B, and C boxes, and the DNA-binding domain are indicated. The activation domain is enlarged to depict the series of mutant proteins. Arrowheads indicate residues substituted with alanine in each mutant protein (M1, residues 59–62; M2, residues 63 and 64; M3, residues 69 and 70). Epitopes for the 3 antibodies are indicated. Residues in the epitope for CNL and P-Cdx2-S60 antibodies are underlined in the enlarged activation domain. Epitope for the Cterm antibody is indicated by a dotted line in the Cdx2 diagram. (B) Caco2 cells were transfected with 500 ng of the 10xGal4-TK-luc reporter vector, as well as the different Gal4-Cdx2 expression plasmids and CMV-βgal for normalization of transfection. Relative luciferase activity is shown with Gal4-Cdx2 (15–180) set at 100%. (C) The amounts of Gal4-DBD-Cdx2 expression plasmids used were adjusted to normalize the quantity of Gal4-DBD-Cdx2 fusion protein expression determined by immunoblots to the Gal4-DBD. Detection of the Gal4-DBD-Cdx2 fusion proteins after adjustment. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Cdx2 activation domain is phosphorylated at position serine 60. Gal4-Cdx2 fusion proteins and Cdx2 full-length proteins were labeled with 32P-phosphate in transiently transfected 293T cells. The cells were subsequently lysed and 32P-phosphate-labeled proteins were isolated by immunoprecipitation as described in Materials and Methods. Alkaline phosphatase treatment of the immunoprecipitated Flag-Cdx2 proteins was conducted to remove the labeled phosphate. (A) Gal4-Cdx2 fusion proteins were analyzed after in vivo phospholabeling. Total amount of immunoprecipitated protein was measured by Western blotting. (B) Flag-tagged Cdx2 proteins were analyzed after in vivo phospholabeling with or without alkaline phosphatase treatment. Total amount of immunoprecipitated protein was measured by Western blotting. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 A phosphospecific antibody recognizes phosphorylation of serine 60. (A) Immunoprecipitated Flag-Cdx2 proteins in total fractions of transiently transfected 293T cells were analyzed by Western blotting using Cterm and P-Cdx2-S60 antibodies. Alkaline phosphatase treatment was conducted to remove phosphate. (B) Immunoprecipitated Flag-Cdx2 and Flag-Cdx2 S60A proteins in nuclear fractions of transiently transfected 293T cells were analyzed by Western blotting using Cterm and P-Cdx2-S60 antibodies. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Phosphorylation of the serine 60 is dependent on the MAPK pathway. Phosphorylation of Flag-tagged Cdx2 proteins in transiently transfected HCT116 cells was inhibited using increasing concentrations of the (A) MEK1/2 inhibitor UO126 and (B) the MEK1 inhibitor PD98059, respectively. Proteins were immunoprecipitated and analyzed by Western blotting using Cterm and P-Cdx2-S60 antibodies. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Detection of intracellular S60-phosphorylated and S60-nonphosphorylated Cdx2 localization. Transiently transfected HCT116 were screened for the intracellular localization of Cdx2 (A, C, and E) or Cdx2 S60A (B, D, and F) using the CNL (A and B) and the P-Cdx2-S60 (C and D) antibodies in immunocytochemistry, respectively. Rabbit IgG was used as a control (E and F). Original magnification 400×. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Binding of S60-phosphorylated and S60-nonphosphorylated Cdx2 to the Cdx2-specific SIF1 DNA sequence. Nuclear extracts from HCT116 cells transfected with Cdx2 (lanes 2–5) or Cdx2 S60A (lanes 6–9) were incubated with a 32P-labeled SIF1 oligonucleotide (lane 1, probe alone). Complex A (Cdx2 monomers) and B (Cdx2 dimers) were formed. Complexes were supershifted (arrows) with the Cdx2-Cterm (*lanes 3 and 7), P-Cdx2-S60 (**lanes 4 and 8), and CNL antibodies (***lanes 5 and 9), respectively. FP indicates the free probe. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of phosphorylation of the serine 60 residue on the transactivating capacity of Cdx2. (A) Detection of immunoprecipitated Flag-tagged Cdx2 in transiently transfected Caco2 or HCT116 cells using Cterm and P-Cdx2-S60 antibodies in Western blotting analysis, respectively. (B) Caco2 or HCT116 cells were transfected in parallel with 500 ng of the 10xGal4-TK-luc reporter vector, as well as the different Gal4-DBD-Cdx2 expression plasmids. A master mix of DNA and lipofectamine was divided equally over the 2 cell lines to establish equal transfection conditions. Renilla luciferase was used for normalization of transfection. The amounts of Gal4-Cdx2 expression plasmids used were adjusted to normalize the quantity of Gal4-Cdx2 protein expression determined by immunoblots to the Gal4 domain (data not shown). Relative luciferase activity is shown with Gal4-Cdx2 (15–180) set at 100%. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 S60-phosphorylation of Cdx2 is lost during Caco2 cell differentiation. Caco2 cells were analyzed at 2 days preconfluency (-2), confluency (0), 7 and 14 days postconfluency. ColoDM cells were analyzed at confluency and were used as control. Total cell lysates were used for immunoblot analysis, and 50 μg of protein was loaded in each lane. Constant amounts of Cdx2 were detected during Caco2 cell differentiation. Moderate levels of Cdx2 S60-phosphorylation were detected before confluency (lane 1). No phosphorylation of the S60 position was detected after cells reached confluency (lanes 2–4). ColoDM cells, which express Cdx2 endogenously, phosphorylate the S60 position markedly (lane 5). This cell line does not express sucrase-isomaltase endogenously.43 Increasing levels of sucrase-isomaltase mRNA were detected during differentiation of Caco2 cells using RNase protection assay. Levels of 36B4 mRNA remained constant. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 10 Detection of Cdx2 in the adult small intestine. Immunohistochemistry was conducted to detect Cdx2 in adult mouse jejunum, using the P-Cdx2-S60, CNL, and Cterm antibodies, respectively. (A) Cdx2 is predominantly detected in the crypt and lower part of the villus of the small intestine (arrow), using the P-Cdx2-S60 antibody and concomitant developing with DAB (brown nuclei). (B) Using the CNL antibody and NBT/BCIP developing, Cdx2 was primarily detected in villous epithelial cells (blue nuclei). (C) Cdx2 protein is detected in both crypts and villi, up to the tip of the villus, using the Cterm antibody and NBT/BCIP developing (blue nuclei). Original magnification 200×. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 11 Detection of Cdx2 in the adult proximal colon. Immunohistochemistry was conducted to detect Cdx2 in adult mouse proximal colon, using the P-Cdx2-S60 and the Cterm antibodies with DAB developing, respectively. (A) Using P-Cdx2-S60, Cdx2 is detected in most nuclei along the epithelial lining of the crypts, with increased signal in the bottom of the crypts (arrow). (B) Using Cterm, staining is equally found along the crypts. (C) Alkaline phosphatase treatment of the tissues eliminated the recognition of the epitope for the P-Cdx2-S60 antibody completely. (D) Alkaline phosphatase treatment of the tissues did not affect staining with Cterm antibody. Original magnification 200×. Gastroenterology 2001 121, 1437-1450DOI: (10.1053/gast.2001.29618) Copyright © 2001 American Gastroenterological Association Terms and Conditions