Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells.
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.
Lopez-Ilasaca
Marco A
MA
Cardiovascular Research Laboratories, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Bernabe-Ortiz
Julio C
JC
Na
Soon-Young
SY
Dzau
Victor J
VJ
Xavier
Ramnik J
RJ
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
D048868:Adaptor Proteins, Signal Transducing; C479421:CNKSR2 protein, human