Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton.
Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.
Banerjee
Jayashree
J
Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Wedegaertner
Philip B
PB
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D019556:COS Cells; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D017470:Receptors, Glutamate / Q000737:chemistry* / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014162:Transfection
C577234:ARHGEF11 protein, human; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D020662:Guanine Nucleotide Exchange Factors; D017470:Receptors, Glutamate; D064067:Rho Guanine Nucleotide Exchange Factors; D013844:Thiazoles; D053778:Thiazolidines; D008156:Luciferases; C037068:latrunculin B; D000409:Alanine