Interrogating signaling pathways activation and deactivation with homogeneous NanoBiT Cell based kinase immunoassays

Abstract

Cellular responses such as gene expression, enzyme activities, protein synthesis and translocation are orchestrated through activation of diverse cell signaling pathways. In these pathways, phosphorylation of specific proteins by specific kinases constitute important nodes by which the signal is transduced from an upstream activation event to downstream cellular responses. As a corollary, alteration of these kinase activities can have a detrimental effect on these cellular processes, leading to serious pathologies such as cancer, inflammation and diabetes. Therefore, monitoring these signaling events through the analysis of specific node phosphorylation states can help better understand cell behavior in health and disease states. We developed the NanoBiT cell based kinase immunoassay platform which is a suite of simple homogeneous assays to detect phosphorylation of endogenous proteins of interest in lysed cells. NanoLuc Binary Technology (NanoBiT) is a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments. A cell lysate containing the protein target is incubated with two primary antibodies that recognize separate epitopes on the protein. These 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 the target protein phosphorylation. Unlike current assays such as Western blot or ELISA, that are tedious, require multiple washing steps, and not easily adaptable to HTS, the NanoBiT immunoassay method presented here takes less than two hours to complete in a homogeneous “Add and Read” format. Other advantages include no cell engineering as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. We tested this system by monitoring the activation of multiple signaling pathways including NF‐κB, mTOR/AKT and JAK/STAT, through specific nodes of phosphorylation such as pIκB, pAKT, and pSTAT3. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, mTOR, or JAKs) and obtained the expected pharmacology. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.