Category: Cellular Technologies
Signaling pathways activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential to better understand normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, non-homogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large subunits (11 aa peptide and 18 kDa polypeptide). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (BTK, NF-kB and JAK/STAT). In very few steps we detected the expected biological response of these pathways either by monitoring BTK autophosphorylation, assessing phosphorylation and degradation of IkB or phosphorylation of STAT3 upon activation of the cells with TNFα, or IL6, respectively. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (BTK, IKK, or JAKs) and obtained the expected pharmacology. The bioluminescent system presented here have many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.
Hicham Zegzouti– Senior Research Scientist, Promega Corporation, Madison, WI
Senior Research Scientist
Hicham Zegzouti is a Senior Research Scientist in the Assay Design Division of Research & Development at Promega Corporation. He is interested in the molecular and biochemical processes that regulate cellular signaling and cell-cell communications. His group develops Assay technologies to interrogate diverse enzyme activities and cellular pathways such as kinases, glycosyltransferases, epigenetic enzymes and other drug targets.