Related Experiment Videos
Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition
1Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston 77030, USA.
Abstract:
Decreased expression of C-CAM, a member of the CEA family of immunoglobulin like cell adhesion molecules, occurs in carcinomas of the colon, liver and prostate. Down regulation of C-CAM during the early stages of carcinogenesis in rat liver and human prostate has also been reported. We have recently shown that restoration of the expression of the isoform with long cytoplasmic domain, C-CAM1, leads to suppression of the tumorigenicity of prostatic carcinoma cells in vivo and growth suppression in vitro. These observations suggest that C-CAM1 may play an important role in regulating cell growth in normal tissues. Previous studies have demonstrated that the function of many members of the Ig-supergene family is dependent on interactions with cytoplasmic proteins. In the present study, we have used a bifunctional cross-linker to identify cellular proteins that interact directly with C-CAM1. Immunoblot analysis of WGA bound membrane proteins crosslinked with DSS identified a 180 kDa complex composed of C-CAM and an 80 kDa protein designated CAP-80 (C-CAM Associated Protein). Immunoprecipitation with anti-C-CAM antibodies showed that CAP-80 was co-precipitated with C-CAM from detergent solubilized, WGA-purified proteins. To assess the specificity of CAP-80 binding, the ability of CAP-80 to form stable complexes with C-CAM1 mutants expressed in insect cells was tested. Deletion of the cytoplasmic domain of C-CAM1 abolished complex formation whereas deletion of the extracellular Ig domains had no effect. These results suggest that a CAP-80 homologue (ICAP-80) is present in insect cells and ICAP-80 interacts with the cytoplasmic domain of C-CAM1. Replacement of Tyr488, a residue in the cytoplasmic domain known to be phosphorylated in vivo, with Phe did not diminish the association between C-CAM1 and ICAP-80, suggesting that Tyr488 phosphorylation is not required for association. The ability of various C-CAM1 mutants to associate with ICAP-80 correlated with their growth inhibitory activities, suggesting that ICAP-80/CAP-80 may play an important role in C-CAM1-mediated growth inhibition.
Insights
Cell adhesion molecule C-CAM1, when its expression is restored, suppresses tumor growth. Researchers identified CAP-80 as a protein interacting with C-CAM1's cytoplasmic domain, crucial for this growth inhibition.
Area of Science:
- Cell adhesion molecules
- Cancer biology
- Molecular interactions
Background:
- Decreased expression of cell adhesion molecule C-CAM occurs in various carcinomas.
- Restoring C-CAM1 expression suppresses prostatic carcinoma cell tumorigenicity and growth.
- C-CAM1's function may involve interactions with cytoplasmic proteins.
Purpose of the Study:
- To identify cellular proteins directly interacting with C-CAM1.
- To investigate the role of these interactions in C-CAM1-mediated growth inhibition.
Main Methods:
- Used a bifunctional cross-linker (DSS) to identify interacting proteins.
- Immunoblot analysis and immunoprecipitation to detect C-CAM-associated proteins.
- Tested C-CAM1 mutants in insect cells to assess binding specificity.
Main Results:
- Identified an 80 kDa protein, CAP-80, that forms a complex with C-CAM1.
- CAP-80/ICAP-80 binding requires C-CAM1's cytoplasmic domain but not Tyr488 phosphorylation.
- The ability of C-CAM1 mutants to associate with ICAP-80 correlated with growth inhibitory activity.
Conclusions:
- CAP-80/ICAP-80 interacts with the C-CAM1 cytoplasmic domain.
- This interaction is critical for C-CAM1-mediated growth inhibition.
- C-CAM1 and its associated proteins represent a potential target for cancer therapy.