Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells

• Published in: FEBS letters Vol. 579; no. 3; pp. 648 - 654
• Main Authors: Lopez-Ilasaca, Marco A., Bernabe-Ortiz, Julio C., Na, Soon-Young, Dzau, Victor J., Xavier, Ramnik J.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 31.01.2005
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Animals; Bioluminescence resonance energy transfer; BRET; Cell Line; Cells, Cultured; CNK; connector enhancer of KSR; Energy Transfer; hCNK1; Humans; Immunoprecipitation; Luminescent Measurements; MAPK; Mice; Microscopy, Fluorescence; mitogen-activated protein kinase; paraformaldehyde; PBS; PFA; phosphate buffered solution; Ras; Rho; Serum response element; Signal Transduction; SRE; hCNK1; BRET; PBS; Signal transduction; CNK; Ras; SRE; Rho; PFA; Serum response element; MAPK; Bioluminescence resonance energy transfer
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2004.12.039

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.