CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis.
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 were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.
Kirshner
Julia
J
Graduate School of the City of Hope and Beckman Research Institute, Duarte, California 91010, USA.
Schumann
Detlef
D
Shively
John E
JE
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology*; D000943:Antigens, Differentiation / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D002460:Cell Line; D045744:Cell Line, Tumor; D003094:Collagen / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D004338:Drug Combinations; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000494:pharmacology; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors
D017306:Annexin A2; D015703:Antigens, CD; D000943:Antigens, Differentiation; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D004338:Drug Combinations; D007797:Laminin; D010455:Peptides; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; C059485:matrigel; D003094:Collagen; D005982:Glutathione Transferase