Carcinoembryonic Antigen Cell Adhesion Molecule 1 Directly Associates with Cytoskeleton Proteins Actin and Tropomyosin

Abstract

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 cellcell 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 Lisoform (GST-Cyto-L) bound poorly to F-actin in a cosedimentation assay, the S-isoform fusion protein (GSTCyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K D ‫؍‬ 7.0 M). 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 ؋ 10 ؊8 and 1.0 ؋ 10 ؊7 M for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 ؋ 10 ؊8 and 1.3 ؋ 10 ؊7 M for GSTCyto-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 ؋ 10 ؊5 and 1.8 ؋ 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. CEACAM1 1 (biliary glycoprotein, CD66a) is a member of the carcinoembryonic antigen (CEA) family, which in turn belongs to the Ig superfamily (1-4). Alternative splicing of the transcripts of a single gene results in expression of at least four CEACAM1 isoforms (5, 6), all of which contain a transmembrane region, followed by a 74-amino acid long (CEACAM1-L) or a 14-amino acid short (CEACAM1-S) cytoplasmic domain. CEACAM1 is a highly glycosylated type 1 transmembrane protein expressed on the surface of epithelial, endothelial, and granulocytic cells (7). The human as well as the rat and mouse isoforms of CEACAM1 have been characterized as homotypic cell adhesion molecules (8, 9). Murine CEACAM1 also functions as a receptor for murine hepatitis virus (10), whereas human CEACAM1 can bind bacterial membrane proteins from Escherichia coli, Salmonella typhimurium, or Neisseria gonorrhoeae (11,12). CEACAM1 expression is down-regulated in human colon (13) and prostate (14) cancer and in 30% of breast cancers (15). Transfection of rat CEACAM1-L into a human tumorig...

The Cell-Cell Adhesion Molecule Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Inhibits IL-2 Production and Proliferation in Human T Cells by Association with Src Homology Protein-1 and Down-Regulates IL-2 Receptor

“…The results for CaM were even more dramatic, with a 5-fold increase of binding after CEACAM1 ligation. The increased association with CaM after CEACAM1 ligation correlates with our previous finding that CaM inhibits the association of CEACAM1 with actin and tropomyosin (12). Because T cell activation involves Vav and cytoskeletal reorganization (34), CEACAM1 may play a role in this pathway too.…”

“…2). We also Western blotted for ZAP-70, a major constituent of the activated TCR complex, and CaM, which we (12) and Obrink and coworkers (29) have previously shown to associate with the cytoplasmic domain of CEACAM1. Low levels of ZAP-70 were found associated with CEACAM1 before and after ligation with T84.1 with a 2-fold increase after ligation.…”

“…Anti-CEACAM1 T84.1 (24) and rabbit Ab 22-9 to the long cytoplasmic domain (12) have been previously described. Anti-CEACAM1 4/3/17 was a gift from F. Grunert (VacGene, Freiburg, Germany) (25,26).…”

“…A critical Ser residue (S-503 in rat CEACAM1-4L) has also been identified in the long cytoplasmic domain that controls signaling via the insulin receptor in rat hepatocytes (9,10). The 14-aa-short cytoplasmic domain lacks Tyr residues, but can be phosphorylated by protein kinase C on Ser and Thr residues (11) and binds calmodulin (CaM) (11) and both actin and tropomyosin (12). Recently, we have shown that CEACAM1-4S reverts breast cancer epithelial cells to a normal morphology and mediates lumen formation in a three-dimensional culture (13).…”

“…Stable cell lines were obtained by transfection with human CEACAM1-4L or -4S (12,13). Expression levels of the two isoforms were equivalent, as measured by FACS and Western blot analysis (data not shown).…”