title: DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA.
abstract: In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.
authors.affiliation: Institute of Genetics, Queen's Medical Centre, School of Biology, The University of Nottingham, Nottingham NG7 2UH, UK.
authors.forename: Konstantinos J
authors.initials: KJ
authors.lastname: Mavrakis
authors.forename: Karen J
authors.initials: KJ
authors.lastname: McKinlay
authors.forename: Peter
authors.initials: P
authors.lastname: Jones
authors.forename: Fred
authors.initials: F
authors.lastname: Sablitzky
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D019556:COS Cells; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D048429:Cell Size / Q000187:drug effects / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020536:Enzyme Activators / Q000302:isolation & purification / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011554:Pseudopodia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D022502:Stress Fibers / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C479750:DEF6 protein, human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020536:Enzyme Activators; D020662:Guanine Nucleotide Exchange Factors; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C491873:SWAP70 protein, human; C113365:Swap70 protein, mouse; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein