Antisem to carcinoembryonic antigen recognizes a phosphotyrosine‐containing protein in human colon cancer cell lines

Human biliary glycoprotein (BGP)1 is a member of the CEA gene family, a gene cluster on chromosome 19, including CEA, BGP, nonspecific cross-reacting antigen, and pregnancy-specific glycoprotein (1-5). BGP was first described as a CEA cross-reacting substance in bile (6) and was localized to the cell surface of bile canaliculi in the liver and gallbladder mucosa (7). Molecular cloning studies on BGP (8, 9) revealed that it exists in alternatively spliced isoforms from a single gene (9). BGP has a transmembrane domain and, depending on alternative splicing, a long (71 amino acids) or short (10 amino acids) cytoplasmic domain. The long form can be phosphorylated by protein kinases (10) after activation by antibody binding (11) We have previously shown that type II interferon, interferon-␥ (IFN-␥), is capable of up-regulating both the BGP and CEA genes, but by different mechanisms (22). CEA is slowly induced by IFN-␥ over a period of 24 -72 h, requiring new protein synthesis, while BGP is rapidly induced from 4 -24 h, without significant inhibition by cycloheximide (22). Rapid induction of gene expression by IFN-␥ is known to be mediated by GAS (for a review, see Ref. 23), found in a variety of genes, including interferon regulatory factor-1 (IRF-1). IRF-1, in turn, can activate a large number of genes by binding to the interferon-stimulated response element (ISRE). ISRE was previously identified for genes activated by type I interferons, IFN-␣ and IFN-␤. Thus, gene activation by IRF-1 is a major crossover pathway for gene activation by type I and II interferons. The mRNA for IRF-1 has a short half-life (30 min) (24) and is rapidly down-regulated by IRF-2 by competing for the same ISRE-binding site (25,26). Because of their potent effects on cell growth, IRF-1 has been termed a tumor suppressor gene and IRF-2 an oncogene (27).In this report, we show that the BGP promoter has an ISRE that is specifically activated by IRF-1 after treatment of colorectal or HeLa (cervical) cells with IFN-␥. IRF-1 mRNA is superinduced in these cells by cycloheximide, and a major species of BGP mRNA is inhibited by cycloheximide. We also show that both USF and Sp1 bind to another footprint in the BGP promoter and that an Sp1-like protein is induced by IFN-␥ in HT-29 cells. MATERIALS AND METHODS Cell Culture and TreatmentThe colon carcinoma cell lines HT-29 and SW403 and the cervix carcinoma cell line HeLa were obtained from American Type Culture Collection. The cells (0.5 ϫ 10 6 cells/ml) were suspended in 20 ml of medium in 75-cm 2 flasks, allowed to reach semiconfluency (3 days), and

Section: Discussionmentioning

confidence: 99%

Section: Human Biliary Glycoprotein (Bgp)mentioning

confidence: 99%

Role of Interferon Regulatory Factor-1 in the Induction of Biliary Glycoprotein (Cell CAM-1) by Interferon-γ

Chen

Lin

Shively

1996

“…In this regard, members of the carcino embryonic antigen-related glycoprotein family have been reported to associate with molecules that could feed into downstream MAP kinases. Tyrosine-phosphorylated biliary glycoprotein can reversibly associate with the protein tyrosine phosphatase SHP-1 (38), whereas CD66a, a human homologue, can be tyrosine-phosphorylated and associate with pp60 c-src , leading to increased c-Src activity in vitro (39,40). Activated c-Src is a classic inducer of the Ras and Raf proteins, both potentially upstream of ERK (41,42), and it is clear that tyrosine phosphatase activity can regulate both the ERK and p38 MAP kinases (43)(44)(45)(46)(47)).…”

Section: Figmentioning

confidence: 99%

Murine Hepatitis Virus Strain 3 Induces the Macrophage Prothrombinase fgl-2 through p38 Mitogen-activated Protein Kinase Activation

McGilvray

Wei

et al. 1998

The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase, fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitro kinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosinephosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1-5 min, ERK phosphorylation and activity increase more gradually, peaking at 20 -30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1-20 M) abolished the virally stimulated induction of fgl-2 mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1-50 M) limited fgl-2 functional activity but had little to no effect on fgl-2 mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage both in vitro and in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2 expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis.

“…To understand the biological function of the two isoforms, studies have focused on the identification of cellular proteins interacting with the cytoplasmic domain of CEACAM1-L. CEACAM1-L has been shown to be phosphorylated in vitro on its tyrosine as well as its serine and threonine residues (19), in neutrophils after antibody binding (20), or in hepatocytes after activation of the insulin receptor (21,22). The reported association of human CEACAM1-L or its murine and rat homologues with Src family kinases (23,24) as well as the protein-tyrosine phosphatases SHP-1 (25) and SHP-2 (26) suggests a signal transduction role for CEACAM1-L.…”

Section: Ceacam1mentioning

confidence: 99%

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

Schumann¹,

Chen²,

Kaplan³

et al. 2001

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...