The Rho Guanine Nucleotide Exchange Factor Lsc Homo-oligomerizes and Is Negatively Regulated through Domains in Its Carboxyl Terminus That Are Absent in Novel Splenic Isoforms

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (33), p.30975-30984
• Main Authors: Eisenhaure, Thomas M., Francis, Sanjeev A., Willison, L.David, Coughlin, Shaun R., Lerner, Daniel J.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; COS Cells; Guanine Nucleotide Exchange Factors; Isomerism; Mammals; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis; Protein Structure, Tertiary; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; rho GTP-Binding Proteins - metabolism; Rho Guanine Nucleotide Exchange Factors; Spleen - metabolism; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M303277200

Rho GTPases control fundamental cellular processes, including cytoskeletal reorganization and transcription. Rho guanine nucleotide exchange factors (GEFs) compose a large (>65) and diverse family of related proteins that activate Rho GTPases. Lsc/p115-RhoGEF is a Rho-specific GEF required for normal B and T lymphocyte function. Despite its essential role in lymphocytes, Lsc/p115-RhoGEF signaling in vivo is not well understood. To define Lsc/p115-RhoGEF signaling pathways in vivo, we set out to identify proteins that interact with regulatory regions of Lsc. The 146-amino acid C terminus of Lsc contains a predicted coiled-coil domain, and we demonstrated that deletion of this C terminus confers a gain of function in vivo. Surprisingly, a yeast two-hybrid screen for proteins that interact with this regulatory C terminus isolated a larger C-terminal fragment of Lsc itself. Co-immunoprecipitation experiments in mammalian cells demonstrated that Lsc specifically homo-oligomerizes and that the coiled-coil domain in the C terminus is required for homo-oligomerization. Mutagenesis experiments revealed that homo-oligomerization and negative regulation are distinct functions of the C terminus. Two novel isoforms of Lsc found in the spleen lack portions of this C terminus, including the coiled-coil domain. Importantly, the C termini of both isoforms confer a gain of function and eliminate homo-oligomerization. These results define two important features of Lsc signaling. First, Lsc homo-oligomerizes and is negatively regulated through domains in its C terminus; and second, functionally distinct isoforms of Lsc lacking these domains are present in the spleen.