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.
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.
Eisenhaure
Thomas M
TM
Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA.
Francis
Sanjeev A
SA
Willison
L David
LD
Coughlin
Shaun R
SR
Lerner
Daniel J
DJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020662:Guanine Nucleotide Exchange Factors; D007536:Isomerism; D008322:Mammals; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D013154:Spleen / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins / Q000378:metabolism
C577231:Arhgef1 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins