| The mechanism by which T lymphocytes become activated during an immune response is complex. One important component of T lymphocyte activation is the CARMA1 protein, which plays an essential role in T cell receptor/CD28 costimulation-induced NF-kappaB activation. The CARMA1 protein is composed of a Caspase recruitment domain (CARD), a Coiled-coil domain (CC), as well as a membrane-associated guanylate kinase domain (MAGUK). The objective of this research was to investigate the mechanism by which CARMA1 localizes to the cell membrane, and to uncover novel CARMA1-associated proteins. A series of Yeast Two Hybrid (Y2H) screenings to uncover novel protein-protein interactions were undertaken. A Y2H screen with the N-terminal domains of CARMA1, CARD+CC, revealed an interaction with a truncated CARMA1 protein. Further analysis by co-immunoprecipitation assays demonstrated that CARMA1 can oligomerize through its CC domain, and that oligomerization is independent of cell activation. The relevance of this oligomerization to T cell activation was assessed utilizing oligomerization-deficient CARMA1 mutants, which disrupt physical, functional, and recruitment aspects of CARMA1 signaling. A Y2H screen with the MAGUK domain of CARMA1 revealed a novel binding partner, the Calcium and Integrin Binding protein (CIB1). Physical interaction analysis demonstrates that CIB1 associates with CARMA1 but not with a functionally defective mutant CARMA1 L808P. It was also found that CARMA1 and CIB1 are constitutively associated. While present data suggest a role of the CIB1 protein in TCR signaling, ongoing experimentation is necessary to determine a functional relevance for the CARMA1-CIB1 interaction. |
