Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Abstract

CEACAM1-4S (carcinoembryonic antigen cell CEACAM14 (carcinoembryonic antigen cell adhesion molecule 1) is a type I membrane glycoprotein that mediates homotypic cell adhesion and belongs to the CEA gene family (1). It has been shown to play a role in bacterial and viral uptake (2-5), in insulin clearance (6), angiogenesis (7), NK and T-cell regulation (8 -11), and tumor suppression (12, 13). Despite its proposed function as a cell-cell adhesion molecule, CEACAM1 is often found on the lumenal surface of epithelial cells, suggesting the possibility that it also plays a role in cell polarization and lumen formation. In this respect, we have recently shown that CEACAM1 is expressed on the lumenal surface of normal mammary epithelial cells (14), and that in a three-dimensional model of mammary morphogenesis, blocking its expression with either an antisense gene or anti-CEACAM1 antibodies blocks lumen formation (15, 16), while breast cancer cells, which lack CEACAM1 and fail to form lumena in a three-dimensional culture, are restored to normal when transfected with the epithelial specific CEACAM1-4S isoform (16). CEACAM1 gene expression has been shown to be down-regulated early in colon cancer (12,17), and in a variety of cancer models, re-introduction of the CEACAM1 gene causes reversion of the malignant phenotype (18 -20). Although CEACAM1 is expressed in multiple isoforms with 1-4 Ig-like ectodomains and either long (72 amino acids) or short (12-14 amino acids) cytoplasmic domains, the short cytoplasmic domain isoforms predominate in epithelial cells. Because the cytoplasmic domain of this isoform is only 12-14 amino acids in length (the exact start of the domain is in doubt) it was originally thought not to play a role in signal transduction, but perhaps acted in a dominant negative fashion in the presence of the long cytoplasmic domain. However, we have shown that peptides or glutathione S-transferase fusion proteins from the short cytoplasmic domain bind directly to actin, tropomyosin, calmodulin, and more recently, to the annexin-2 p11 tetramer AIIt (21-23).In this study, we have used mutational analysis of the short cytoplasmic domain of CEACAM1 to reveal which residues are involved in actin interactions and lumen formation. The results of our studies show that mutants F454A and K456A greatly decrease or increase, respectively, actin interactions, whereas the F454A and the double mutant T457A,S459A greatly reduce lumen formation. Further analysis reveals that either Thr 457 or Ser 459 are phosphorylated during lumen formation. The studies that identified these key residues are the subject of this report. MATERIALS AND METHODS Construction of Baits for Yeast Two-hybrid ScreeningThe nucleotide and amino acid sequence for CEACAM1-4S is taken from NCBI entry number NM_001712, gi:4502404, the full-length coding sequence including the N-terminal signal * This work was supported by National Institutes of Health NCI Grant CA84202. The costs of publication of this article were defrayed in part by the payment of page ch...

Induction of Lumen Formation in a Three-dimensional Model of Mammary Morphogenesis by Transcriptional Regulator ID4

“…Necessary and Essential for Lumen Formation-As previously reported, CEACAM1-SF restored lumen formation to MCF7 cells grown in a three-dimensional culture, whereas a functionally null, phosphorylation site mutant in the cytoplasmic domain of CEACAM1-SF failed to form lumena (7). A gene chip analysis comparing gene expression of CEACAM1-wild type (SW) transfected MCF7 cells and the functionally null, double alanine mutant (DA) MCF7 cells, indicated among other genes, that the ID4 gene was up-regulated by a log2 of 6.7-fold (8).…”

“…murine model of lumenogenesis (26). Thus, restoration of CEACAM1 expression is able to trigger a normal tissue pathway in a breast cancer cell line in both in vitro (7) and in vivo models (26). In addition, mutation of two residues (Thr-457 and Ser-459) to alanine (double alanine or DA mutant) in the cytoplasmic domain of the short isoform abrogated its lumen formation function (7).…”

“…Thus, restoration of CEACAM1 expression is able to trigger a normal tissue pathway in a breast cancer cell line in both in vitro (7) and in vivo models (26). In addition, mutation of two residues (Thr-457 and Ser-459) to alanine (double alanine or DA mutant) in the cytoplasmic domain of the short isoform abrogated its lumen formation function (7). Among the various up-regulated genes that were differentially expressed between the short isoform, wild type CEACAM1-transfected MCF7 cells (SW) and the double mutant, nonlumen forming CEACAM1-transfected cells (DA), ID4 was identified for further study due to its postulated role as a tumor suppressor in several cancers, including breast cancer (9).…”

“…Transfection of cells was performed at 50% confluence in T25 flasks overnight prior to being transfected with pCMV6-Vector or CaMK2D or ID4 plasmids (Origene) using the Cell Line Nucleofector Kit V on the Nucleofector 2b Device (Lonza, Walkersville, MD). The three-dimensional Matrigel (BD Biosciences) sandwich assay has been previously described (7). Briefly, 10 ml of culture dishes were coated with 1 ml of Matrigel and incubated at 37°C for 30 min until the Matrigel solidified, and cells (1 ϫ 10 6 ) in 10 ml of mammary epithelial basal medium plus pituitary gland extract (Lonza Group LTD., Switzerland) were added to each dish.…”

“…As a next step in identifying factors downstream from CEACAM1 in lumenogenesis, we identified key residues in the short cytoplasmic domain isoform (CEACAM1-SF), the predominant isoform in breast that comprised a short stretch of 12 amino acids (7). Because CEACAM1 can be expressed as multiple isoforms, depending on its tissue of origin, we refer to the cytoplasmic isoform expressed exclusively in the mammary gland as the short form, or CEACAM1-SF (referring only to the cytoplasmic domain).…”