title: The RhoA-binding protein, rhophilin-2, regulates actin cytoskeleton organization.
abstract: The Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP- and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rho-induced serum response element transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2-expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active Rho mutant reversed the disassembly phenotype, in which the coexpressing cells were more spread and less contracted than Rho alone-expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity.
authors.affiliation: Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Peck
authors.forename: Michael
authors.initials: M
authors.lastname: Oberst
authors.forename: Kerrie B
authors.initials: KB
authors.lastname: Bouker
authors.forename: Emma
authors.initials: E
authors.lastname: Bowden
authors.forename: Peter D
authors.initials: PD
authors.lastname: Burbelo
mesh_terms: D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014018:Tissue Distribution; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D020033:Protein Isoforms; C471419:RHPN2 protein, human; D011993:Recombinant Fusion Proteins; C097916:rhophilin; D008156:Luciferases; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D020742:rhoA GTP-Binding Protein