BRET assays have a number of advantages in comparison to more conventional approaches such as immunoprecipitation or yeast two-hybrid systems specifically, BRET is quantitative and allows in vivo detection of molecular interactions in a native cellular environment with post-transcriptional modifications and is unaffected by small variations in expression levels. BRET assays have been used recently in the discovery of a number of novel interactions between G protein-coupled receptors, the characterization of integrins and TGFβ receptor interaction or dimerization of STAT3. This protein–protein interaction assay is based on energy transfer from a bioluminescent donor (Luciferase) to a fluorescent acceptor protein (GFP). In order to test these putative interactions in a systematic way using intact cells, we established bioluminescence resonance energy transfer (BRET) assays. Since CNK proteins have been proposed to act as scaffolds in the Ras-MAPK pathway, we sought to explore the possibility that CNK1 could interact with members of the small GTPase family of Ras and/or Rho proteins in vivo. It is not known whether hCNK1 also binds other proteins in the Ras signaling pathway and the biochemical relationship of hCNK1 to small GTPases is unknown. dCNK1 interacts with Raf through its C-terminal portion and its expression is required for insulin-dependent Raf activation. Structural comparisons of dCNK have identified three separate genes in humans: CNK1 and CNK2 share conserved domain structure with dCNK two separate genes on chromosome 6, CNK3A and CNK3B share only partial domain homology with Drosophila CNK. By virtue of its modular primary structure, CNK potentially could couple multiple effector responses these domains include a sterile alpha motif (SAM) domain, a PDZ domain, two proline-rich (potential SH3-binding) domains, and a C-terminal pleckstrin homology (PH) domain such domains are found in many proteins involved in signaling and suggest further interactions of CNK with other proteins and small molecules. dCNK was found to cooperate strongly with activated RAS when coexpressed in the Drosophila eye and seems to be required for normal cell proliferation and differentiation. The dCNK protein (named for connector enhancer of KSR) was identified as an enhancer of a Drosophila dominant-negative KSR mutant. elegans has shown the existence of a number of scaffold proteins such as KSR1, dCNK, Sur-8, or MP1, to play a role in Ras-MAPK pathway. Īlthough the Ras/Rho signaling has been extensively studied in a number of systems, our understanding of how signals are regulated through these small GTPases is still far from complete. Members of the subfamily of Rho proteins (Rho, Rac and Cdc42) control intracellular signaling processes through binding to a number of effector proteins such as the WASP family of proteins, PAK, PAR 6, among others, that in turn modulate the actin cytoskeleton, transcriptional outputs, cell proliferation and differentiation. Among the best characterized effectors of Ras proteins are the Raf protein kinase family, the phosphatidylinositol 3-kinase and the Ral-GDS exchange factor for the small GTPase Ral, which control the activation of the mitogen-activated protein kinase (MAPK) pathway, actin cytoskeleton, vesicular trafficking and play a major role in controlling both cell proliferation and differentiation. Ras and Rho proteins control these pathways by binding to effector proteins through its effector loop regions, which are thought to activate specific signaling cascades.
The Ras and Rho family of small GTPases play a critical role in transducing extracellular signals controlling cell proliferation, differentiation and survival.
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