Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin.
CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cell-cell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the l-isoform (GST-Cyto-L) bound poorly to F-actin in a co-sedimentation assay, the S-isoform fusion protein (GST-Cyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K(D) = 7.0 microm). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 x 10(-8) and 1.0 x 10(-7) m for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 x 10(-8) and 1.3 x 10(-7) m for GST-Cyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K(D) values of 1.3 x 10(-5) and 1.8 x 10(-5) m, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin.
Schumann
D
D
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Chen
C J
CJ
Kaplan
B
B
Shively
J E
JE
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D000943:Antigens, Differentiation / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D002272:Carcinoembryonic Antigen; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D000199:Actins; D000596:Amino Acids; D015703:Antigens, CD; D000943:Antigens, Differentiation; C069847:CD66 antigens; D002272:Carcinoembryonic Antigen; C486183:Ceacam1 protein, mouse; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; C076715:pervanadate; D014638:Vanadates; D005982:Glutathione Transferase