ABSTRACT
Engulfment of foreign pathogens is an evolutionary ancient host cell endocytic response. Signaling pathways effecting phagocytosis are divergent and largely depend on the structural features of the cell surface receptor utilized. CEACAM3, a member of the CD66 complex on human neutrophils, has been implicated as a cellular receptor promoting phagocytosis of microorganisms. The cytoplasmic domain of CEACAM3 (CEACAM3(cyt)) contains an immunoreceptor tyrosine-based activation motif. In this study we demonstrate that CEACAM3(cyt) is phosphorylated by protein kinase C, casein kinase I, and Src-kinase in vitro. To identify molecules binding to CEACAM3(cyt) in vivo, we used differentially phosphorylated recombinant expressed CEACAM cytoplasmic domains to isolate CEACAM3(cyt)-associated proteins from granulocyte extracts. Calprotectin, which modulates neutrophil integrin-mediated adhesion and leukocyte trafficking and displays antimicrobial activity, interacts specifically with CEACAM3(cyt). This interaction is calcium-modulated but independent of phosphorylation of CEACAM3(cyt). Although tyrosine-phosphorylated CEACAM3(cyt) binds and stimulates Src-kinases in vitro, no CEACAM3(cyt)-associated phosphokinase activity was copurified.
Subject(s)
Carcinoembryonic Antigen/metabolism , Granulocytes/microbiology , Granulocytes/physiology , Membrane Glycoproteins/metabolism , Neural Cell Adhesion Molecules/metabolism , Calcium Signaling , Carcinoembryonic Antigen/chemistry , Carcinoembryonic Antigen/genetics , Casein Kinases , Humans , In Vitro Techniques , Leukocyte L1 Antigen Complex , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/genetics , Phagocytosis , Phosphorylation , Protein Kinase C/metabolism , Protein Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , src-Family Kinases/metabolismABSTRACT
CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin beta(3) as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin beta(3) interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin beta(3) at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin beta(3) complexes in cell invasion.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation/metabolism , Colonic Neoplasms/pathology , Melanoma/pathology , Platelet Membrane Glycoproteins/metabolism , Antigens, CD/chemistry , Antigens, CD/isolation & purification , Antigens, Differentiation/chemistry , Antigens, Differentiation/isolation & purification , Binding Sites , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/isolation & purification , Cell Adhesion Molecules/metabolism , Chromatography, Affinity , Endometrium/cytology , Female , Granulocytes/physiology , Humans , Integrin beta3 , Microscopy, Confocal , Phosphorylation , Platelet Membrane Glycoproteins/chemistry , Platelet Membrane Glycoproteins/isolation & purification , Pregnancy , Pregnancy Trimester, First , Tumor Cells, Cultured , TyrosineABSTRACT
CEACAM1 functions as an epithelial tumor suppressor and as an angiogenic growth factor. In the present study, utilizing differentially (serine/threonine or tyrosine) phosphorylated cytoplasmic domains of CEACAM1 and CEACAM3 as bait to isolate associated proteins from granulocyte extracts, we have identified human paxillin as a binding partner of the tyrosine-phosphorylated cytoplasmic CEACAM1 domain. CEACAM1-paxillin complexes were coimmunoprecipitated from extracts of granulocytes, the colonic cell line HT29, and HUVECs. We identified phosphorylated Tyr-488-a residue in the cytoplasmic CEACAM1 domain known to be essential for the tumor suppressive effect-to be necessary for this association. The CEACAM1-paxillin interaction was confirmed using laser scanning confocal microscopy analyses in granulocytes and HT29 cells, where CEACAM1 colocalizes with paxillin at the plasma membrane. In HUVECs a highly polarized expression pattern and colocalization of paxillin and CEACAM1 was observed. These findings support the findings that CEACAM1 is linked to the actin-based cytoskeleton.
