CEACAM1, a Cell-Cell Adhesion Molecule, Directly Associates with Annexin II in a Three-dimensional Model of Mammary Morphogenesis

Abstract

The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins The was using and II and of CEACAM1 with annexin II in epithelial in CEACAM1 with a of the the and with annexin II in the cytoplasmic domain of to directly with which is to with of using of to the cytoplasmic domain of The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins The was using and II and of CEACAM1 with annexin II in epithelial in CEACAM1 with a of the the and with annexin II in the cytoplasmic domain of to directly with which is to with of using of to the cytoplasmic domain of antigen cell adhesion with Ig-like and a cytoplasmic with Ig-like and a short cytoplasmic cell adhesion with Ig-like and a cytoplasmic with Ig-like and a short cytoplasmic cell adhesion is a of the in is a of the CEACAM1 four that protein with four Ig-like ectodomains and a amino short amino cytoplasmic CEACAM1 was identified in liver and a antigen and was to the of of breast, and and in and the of CEACAM1 is CEACAM1 was identified a adhesion molecule in and CEACAM1 cell adhesion CEACAM1 also and short cytoplasmic of which the form to in the CEACAM1 a in in a of and in in CEACAM1 to down-regulated in liver and The in the and in of of the CEACAM1 cell in CEACAM1 many of the of a the cytoplasmic to in The cytoplasmic domain of CEACAM1 in that and and domain to and to and of with the The short cytoplasmic domain also with and and and is to a in in a of II II, is a and protein with a and a domain with and and a of in and in and is with and annexin II Annexin II and down-regulated in a of liver and II a of the protein annexin II a annexin II and Annexin II is the is the annexin II and to Annexin II and fusion of a in and and is with also to a in and cell Annexin II is to in the of epithelial Annexin II is in the annexin II is in the of is to the and annexin II is with of we identified annexin II protein that with the short cytoplasmic domain of was identified using a glutathione S-transferase pull-down assay and and was to directly the cytoplasmic domain of of the of CEACAM1 with annexin II and of and and a of epithelial and and to was and fusion proteins the amino of the cytoplasmic domain of to the using and and and and and and and fusion proteins using fusion protein to the and the cytoplasmic domain using with to the of a in a to the and amino The a to a the cytoplasmic domain of in a in with of the was with to and the cell with of fusion proteins to of The of fusion proteins was the of the the to the fusion The was incubated a The and with of the The was with and The was with was to the to proteins and with to using and of the with and using and the in a and in of of in a using a The was to using was of with and of The using a to a of The was using The was to a using a and was the to the using a with a The was and in the The and The was with a and with the in cell of and proteins with CEACAM1 and annexin the incubated with II a of the incubated with a and was using the and to the Annexin II was in with pull-down assay of was with of fusion proteins to The was incubated and with of The was with to and with II in using the directly the of a of of protein in of a of the was with of of and of the a of in to and and the was with of of and incubated with II to the incubated with was to was a of Annexin II with The cytoplasmic domain of amino the of the and of the cytoplasmic and that we that of the cell with the the to a in a we that of the with the the in and with the the and the proteins we with a fusion protein of the amino of the cytoplasmic domain of the the and cell incubated with fusion and the proteins with and and the and to with The in was identified annexin II with and of also the annexin II was was in the and a in the the annexin II identified of in a that annexin II was in the a the proteins with II a annexin II was in the pull-down the the fusion the of annexin II to the cytoplasmic we also and with the the the of annexin II with that of annexin II of the and with the that of the cytoplasmic domain a in annexin II is of the the of in with annexin II was cell with and that cell lysates, which with and the with which The with II The that annexin II is with a was with II, annexin II was the of the in the annexin II was with with CEACAM1 CEACAM1 was identified in the with Annexin II and in in the of CEACAM1 with annexin II in a we in of in which the epithelial cell was in the form with in which CEACAM1 is in and to the of the the annexin II, and of the of was the and (14 of CEACAM1 was also to with annexin II in the a of annexin II in CEACAM1 we in the form of annexin II, is to to the CEACAM1 is annexin II is in the II and with CEACAM1 in a epithelial cell in The epithelial cell was in in and annexin II, and CEACAM1 is in annexin II is in and of the is of annexin II and CEACAM1 in in in and annexin II and CEACAM1 CEACAM1 is in annexin II is in and of the is to the of annexin II and CEACAM1 in to directly associates with CEACAM1-4S, we in pull-down assay with which is to the amino and fusion proteins and and with and the was and The in that the cytoplasmic domain of the and of in the fusion proteins in the of the of was The of was with that using cell the in that the the and the the The in the is to the that cell proteins annexin that with the that with proteins to directly with fusion proteins the cytoplasmic domain of was incubated with and fusion and proteins to and with II the of of the cytoplasmic domain of with we was the the fusion proteins to to of the the of The was to of the fusion proteins and a to fusion was to a of to the we a in which to the cytoplasmic domain directly to the of the a the amino of the to the of CEACAM1 and a to the The to the amino of the cytoplasmic domain of and the and the the was the to a the of the was also The the of the that the of to of protein the is to the cytoplasmic domain of was a and was a protein to of was a of the was the of to the was and The of and of with the CEACAM1 the that the was the of of the to the a of the of to the of the the of to the the in the of that the of to with the of cytoplasmic the of the pull-down using cell that in the the that of directly the to the cytoplasmic domain of to the cytoplasmic domain the of and the of a in with a of and and was the the and was and of to the cytoplasmic domain of in a that was the that the cytoplasmic domain of amino that with a of proteins the that the of and in we of and and that the of the of the with of to cell in also that of was the was we that the in with the the in the of the to the of the cytoplasmic domain of proteins with the cytoplasmic we of the to the and cell of cell to with the of the pull-down annexin II was identified a protein the and the also we annexin II the was in the and was the the The of the and of using II pull-down cell that proteins directly with we the pull-down assay with which is to to the with cell lysates, the the and the the the of was the cell proteins that with and CEACAM1 and annexin II annexin II and the of of the proteins in we in which of annexin II, and the we the of to the we the that CEACAM1 with in with of the in that the form of annexin II that with of with the cytoplasmic domain of CEACAM1 was The that and with the fusion proteins that the was that the the that is the fusion proteins with a we to a of was we that a the amino the to the cytoplasmic domain of the a of was the fusion to of to the of to we that we that to the cytoplasmic domain of with to the we to we with of the in to the the the in the that the to in is in to the of of to the in the in pull-down we that in cell the and to identified in a in of and the of the lipid in the and to of and the we to the of in the of in is to lipid and to to the of the lipid the to the and in the the to in of annexin II fusion and that also with the fusion protein that of the of the with we and in of of the and the is that the with annexin II is the of to is with a in the of epithelial to a and of The of with the is to the of CEACAM1 with and CEACAM1 and the in to the the of epithelial to the the to the cytoplasmic domain of CEACAM1 and to lipid we that CEACAM1 is a in of to proteins a of is a of is that CEACAM1 and annexin II down-regulated in many cancers. is that the of CEACAM1 and with cell that the antigen cell adhesion with Ig-like and a cytoplasmic with Ig-like and a short cytoplasmic cell adhesion with Ig-like and a cytoplasmic with Ig-like and a short cytoplasmic cell adhesion is a of the in is a of the CEACAM1 four that protein with four Ig-like ectodomains and a amino short amino cytoplasmic CEACAM1 was identified in liver and a antigen and was to the of of breast, and and in and the of CEACAM1 is CEACAM1 was identified a adhesion molecule in and CEACAM1 cell adhesion CEACAM1 also and short cytoplasmic of which the form to in the CEACAM1 a in in a of and in in CEACAM1 to down-regulated in liver and The in the and in of of the CEACAM1 cell in CEACAM1 many of the of a antigen cell adhesion CEACAM1 with Ig-like and a cytoplasmic CEACAM1 with Ig-like and a short cytoplasmic protein glutathione S-transferase antigen cell adhesion CEACAM1 with Ig-like and a cytoplasmic CEACAM1 with Ig-like and a short cytoplasmic protein glutathione S-transferase the cytoplasmic to in The cytoplasmic domain of CEACAM1 in that and and domain to and to and of with the The short cytoplasmic domain also with and and and is to a in in a of Annexin II II, is a and protein with a and a domain with and and a of in and in and is with and annexin II Annexin II and down-regulated in a of liver and Annexin II a of the protein annexin II a annexin II and Annexin II is the is the annexin II and to Annexin II and fusion of a in and and is with also to a in and cell Annexin II is to in the of epithelial Annexin II is in the annexin II is in the of is to the and annexin II is with of we identified annexin II protein that with the short cytoplasmic domain of was identified using a glutathione S-transferase pull-down assay and and was to directly the cytoplasmic domain of of the of CEACAM1 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with CEACAM1 and annexin the incubated with II a of the incubated with a and was using the and to the Annexin II was in with pull-down assay of was with of fusion proteins to The was incubated and with of The was with to and with II in using the directly the of a of of protein in of a of the was with of of and of the a of in to and and the was with of of and incubated with II to the incubated with was to was a of Annexin II with The cytoplasmic domain of amino the of the and of the cytoplasmic and that we that of the cell with the the to a in a we that of the with the the in and with the the and the proteins we with a fusion protein of the amino of the cytoplasmic domain of the the and cell incubated with fusion and the proteins with and and the and to with The in was identified annexin II with and of also the annexin II was was in the and a in the the annexin II identified of in a that annexin II was in the a the proteins with II a annexin II was in the pull-down the the fusion the of annexin II to the cytoplasmic we also and with the the the of annexin II with that of annexin II of the and with the that of the cytoplasmic domain a in annexin II is of the the of in with annexin II was cell with and that cell lysates, which with and the with which The with II The that annexin II is with a was with II, annexin II was the of the in the annexin II was with with CEACAM1 CEACAM1 was identified in the with Annexin II and in in the of CEACAM1 with annexin II in a we in of in which the epithelial cell was in the form with in which CEACAM1 is in and to the of the the annexin II, and of the of was the and (14 of CEACAM1 was also to with annexin II in the a of annexin II in CEACAM1 we in the form of annexin II, is to to the CEACAM1 is annexin II is in the of annexin II and CEACAM1 in in in and annexin II and CEACAM1 CEACAM1 is in annexin II is in and of the is to the of annexin II and CEACAM1 in to directly associates with CEACAM1-4S, we in pull-down assay with which is to the amino and fusion proteins and and with and the was and The in that the cytoplasmic domain of the and of in the fusion proteins in the of the of was The of was with that using cell the in that the the and the the The in the is to the that cell proteins annexin that with the that with proteins to directly with fusion proteins the cytoplasmic domain of was incubated with and fusion and proteins to and with II the of of the cytoplasmic domain of with we was the the fusion proteins to to of the the of The was to of the fusion proteins and a to fusion was to a of to the we a in which to the cytoplasmic domain directly to the of the a the amino of the to the of CEACAM1 and a to the The to the amino of the cytoplasmic domain of and the and the the was the to a the of the was also The the of the that the of to of protein the is to the cytoplasmic domain of was a and was a protein to of was a of the was the of to the was and The of and of with the CEACAM1 the that the was the of of the to the a of the of to the of the the of to the the in the of that the of to with the of cytoplasmic the of the pull-down using cell that in the the that of directly the to the cytoplasmic domain of to the cytoplasmic domain the of and the of a in with a of and and was the the and was and of to the cytoplasmic domain of in a of Annexin II with The cytoplasmic domain of amino the of the and of the cytoplasmic and that we that of the cell with the the to a in a we that of the with the the in and with the the and the proteins we with a fusion protein of the amino of the cytoplasmic domain of the the and cell incubated with fusion and the proteins with and and the and to with The in was identified annexin II with and of also the annexin II was was in the and a in the the that annexin II was in the a the proteins with II a annexin II was in the pull-down the the fusion the of annexin II to the cytoplasmic we also and with the the the of annexin II with that of annexin II of the and with the that of the cytoplasmic domain a in annexin II is of the the of in The with annexin II was cell with and that cell lysates, which with and the with which The with II The that annexin II is with a was with II, annexin II was the of the in the annexin II was with with CEACAM1 CEACAM1 was identified in the CEACAM1 with Annexin II and in in the of CEACAM1 with annexin II in a we in of in which the epithelial cell was in the form with in which CEACAM1 is in and to the of the the annexin II, and of the of was the and (14 of CEACAM1 was also to with annexin II in the a of annexin II in CEACAM1 we in the form of annexin II, is to to the CEACAM1 is annexin II is in the to directly associates with CEACAM1-4S, we in pull-down assay with which is to the amino and fusion proteins and and with and the was and The in that the cytoplasmic domain of the and of in the fusion proteins in the of the of was The of was with that using cell the in that the the and the the The in the is to the that cell proteins annexin that with the that with proteins to the of of the cytoplasmic domain of with we was the the fusion proteins to to of the the of The was to of the fusion proteins and a to fusion was to a of to the we a in which to the cytoplasmic domain directly to the of the a the amino of the to the of CEACAM1 and a to the The to the amino of the cytoplasmic domain of and the and the the was the to a the of the was also The the of the that the of to of protein the is to the cytoplasmic domain of was a and was a protein to of was a of the was the of to the was and The of and of with the CEACAM1 the that the was the of of the to the a of the of to the of the the of to the the in the of that the of to with the of cytoplasmic the of the pull-down using cell that in the the that of directly the that was the that the cytoplasmic domain of amino that with a of proteins the that the of and in we of and and that the of the of the with of to cell in also that of was the was we that the in with the the in the of the to the of the cytoplasmic domain of proteins with the cytoplasmic we of the to the and cell of cell to with the of the pull-down annexin II was identified a protein the and the also we annexin II the was in the and was the the The of the and of using II pull-down cell that proteins directly with we the pull-down assay with which is to to the with cell lysates, the the and the the the of was the cell proteins that with and CEACAM1 and annexin II annexin II and the of of the proteins in we in which of annexin II, and the we the of to the we the that CEACAM1 with in with of the in that the form of annexin II that with of with the cytoplasmic domain of CEACAM1 was The that and with the fusion proteins that the was that the the that is the fusion proteins with a we to a of was we that a the amino the to the cytoplasmic domain of the a of was the fusion to of to the of to we that we that to the cytoplasmic domain of with to the we to we with of the in to the the the in the that the to in is in to the of of to the in the in pull-down we that in cell the and to identified in a in of and the of the lipid in the and to of and the we to the of in the of in is to lipid and to to the of the lipid the to the and in the the to in of annexin II fusion and that also with the fusion protein that of the of the with we and in of of the and the is that the with annexin II is the of to is with a in the of epithelial to a and of The of with the is to the of CEACAM1 with and CEACAM1 and the in to the the of epithelial to the the to the cytoplasmic domain of CEACAM1 and to lipid we that CEACAM1 is a in of to proteins a of is a of is that CEACAM1 and annexin II down-regulated in many cancers. is that the of CEACAM1 and with cell that the that was the that the cytoplasmic domain of amino that with a of proteins the that the of and in we of and and that the of the of the with of to cell in also that of was the was we that the in with the the in the of the to the of the cytoplasmic domain of proteins with the cytoplasmic we of the to the and cell of cell to with the of the pull-down annexin II was identified a protein the and the also we annexin II the was in the and was the the The of the and of using II pull-down cell that proteins directly with we the pull-down assay with which is to to the with cell lysates, the the and the the the of was the cell proteins that with and CEACAM1 and annexin II annexin II and the of of the proteins in we in which of annexin II, and the we the of to the we the that CEACAM1 with in with of the in that the form of annexin II that with The of with the cytoplasmic domain of CEACAM1 was The that and with the fusion proteins that the was that the the that is the fusion proteins with a we to a of was we that a the amino the to the cytoplasmic domain of the a of was the fusion to of to the of to we that we that to the cytoplasmic domain of with to the we to we with of the in to the the the in the that the to in is in to the of of to the in the in pull-down we that in cell the and to identified in a in of and the of the lipid in the and to of and the we to the of in the of in is to lipid and to to the of the lipid the to the and in the the to in of annexin II fusion and that also with the fusion protein that of the of the with we and in of of the and the is that the with annexin II is the of to is with a in the of epithelial to a and of The of with the is to the of CEACAM1 with and CEACAM1 and the in to the the of epithelial to the the to the cytoplasmic domain of CEACAM1 and to lipid we that CEACAM1 is a in of to proteins a of is a of is that CEACAM1 and annexin II down-regulated in many cancers. is that the of CEACAM1 and with cell that the the of and and in the of