Publikationsserver der RWTH Aachen University
Proteomics based analysis of interactions between CEA cell adhesion molecule 1 (CEACAM1) and intracellular proteins in transfected tumor cells
Abstract
dc:descriptionCEA Cell Adhesion Molecule 1 (CEACAM1), a type 1 transmembrane protein belonging to the CEA gene family, is alternatively spliced to produce four major isoforms with a long (CEACAM1-L) or a short cytoplasmic domain (CEACAM1-S). Immunoprecipitations using the murine adenocarcinoma MC38 cell line transfected with human CEACAM1-L were performed to isolate cytoplasmic proteins binding to the unphosphorylated or tyrosine phosphorylated CEACAM1 long cytoplasmic domain. Mass spectrometric analysis of immunoprecipitates separated on two-dimensional gels revealed actin, tropomyosin, myosin, vimentin and cytokeratin as the major proteins interacting with CEACAM1-L after sodium pervanadate induced tyrosine phosphorylation of CEACAM1-L. Surface plasmon resonance studies were performed to characterize actin and tropomyosin binding to the membrane-proximate region of the CEACAM1-L cytoplasmic domain and to its short splicing variant CEACAM1-S. A CEACAM1-L derived peptide bound F-actin and tropomyosin with KDs of 1.3x10–5 M, and 1.8x10-5 M, respectively, while an equivalent CEACAM1-S peptide bound less than 10% of the long form to F-actin and tropomyosin with a KD <10-4 M. GST-long or short cytoplasmic domain fusion proteins bound actin and tropomyosin with KDs of 3.0x10-8 M and 4.1x10-7 M, respectively, for the long form, and KDs of 6.2x10-8 M and 2.6x10-7 M, respectively, for the short form. Calmodulin, a known CEACAM1 binding protein, or EDTA inhibited binding of the CEACAM1-L peptide and both GST-CEACAM1 fusion proteins to actin, while calmodulin and actin, but not EDTA, stimulated binding of GST-CEACAM1-L to tropomyosin. Confocal microscopy studies showed a sodium pervanadate dependent colocalization of F-actin and CEACAM1-L at cell-cell adhesion sites in CEACAM1-L transfected MC38 cells, confirming the initial immunoprecipitation results. The identification of an association of both CEACAM1 cytoplasmic domains with actin and tropomyosin and its potential regulation by calmodulin and Ca2+ could lead to a better understanding of the mechanisms and the functions of interactions between transmembrane cell adhesion proteins and the cytoskeleton.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2000
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Schumann, Detlef
- Contributors dc:contributor
-
- Kreuzaler, Fritz
Subjects
dc:subject × 8Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng