title: Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells.
abstract: Connector enhancer of KSR (CNK) proteins have been proposed to act as scaffolds in the Ras-MAPK pathway. In this work, using in vivo bioluminescence resonance energy transfer (BRET) assays and in vitro co-immunoprecipitation, we show that hCNK1 interacts with the active form of Rho A (G14V) proteins. The domain of hCNK1 that allows binding to Rho proteins involves the C-terminal PH domain. Overexpression of hCNK1 does not affect the actin cytoskeleton and does not modify the appearance of stress fibers in cells overexpressing a constitutively active form of RhoA. In contrast, hCNK1 was able to significantly decrease the RhoA-induced transcriptional activity of the serum response element (SRE) without effect on the Ras-induced SRE activation. These results identify hCNK1 as a specific partner of Rho proteins both in vitro and in vivo and suggest a role of hCNK1 in the signal transduction of Rho proteins.
authors.affiliation: Cardiovascular Research Laboratories, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Lopez-Ilasaca
authors.forename: Julio C
authors.initials: JC
authors.lastname: Bernabe-Ortiz
authors.forename: Soon-Young
authors.initials: SY
authors.lastname: Na
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Dzau
authors.forename: Ramnik J
authors.initials: RJ
authors.lastname: Xavier
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004735:Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D008163:Luminescent Measurements; D051379:Mice; D008856:Microscopy, Fluorescence; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C479421:CNKSR2 protein, human