kinase signaling pathway LinkLight™ assay
Cellular signal transductions involve highly coordinated protein-protein interactions that are regulated by protein phosphorylation and dephosphorylation. Phosphorylation can induce protein conformational changes, and enables phosphorylated proteins to interact with other proteins and transduce signals. A broad range of cellular activities such as cell proliferation, apoptosis, differentiation, and malignant transformation is linked to phosphorylation-mediated protein-protein interactions. Kinases such as ERK1/2, PI3K, AKT, and Src phosphorylate a large number of protein substrates. Phosphorylated proteins in turn interact with other proteins to transduce signals. Kinases do not function in isolation. Kinase interaction networks determine signaling cascades, and eventually cellular functions.
Signal adaptor proteins such as β-arrestins and 14-3-3 proteins have no intrinsic enzymatic activity but link with other proteins to form signal transduction complexes. They are ubiquitously expressed in various types of tissues and serve as hubs to interact with a large number of proteins. The interactions or formation of the signal complexes facilitate the assembly of large molecular complexes that coordinate responses of multiple signaling pathways to incoming stimuli, allowing signal transduction between different cellular compartments.
Many non-receptor kinases, including ERK1/2, PI3K, AKT, JNK, p38, ASK1, GSK3, and Src family members, have been implicated to interact with signal adaptor proteins. Extracellular signal-regulated kinase 2 (ERK2) is a member of the MAP kinase family and is activated in response to various extracellular stimuli. Upon activation, ERK is phosphorylated and released from the RAF/MEK/ERK signal complex. Phosphorylated ERK2 binds β-arrestins and forms ERK2/ β-arrestin signal complex.
Interaction with signal proteins can activate or inactivate kinases, and affect kinase stability and subcellular location. Kinase activity and signaling pathways are interconnected through phosphorylation-mediated protein-protein interactions. Subcellular locations of kinases and effectors determine downstream signaling specificity.
Cell-based kinase signaling pathway LinkLight assays assess kinase and signal adaptor interactions. For example, we demonstrated that activation of diverse cell surface receptors including receptor tyrosine kinases, cytokine receptors, and G-protein coupled receptors by their corresponding ligands produced sensitive ERK2 and β-arrestin interaction signals. Thus, ERK2 and signal adaptor complex acts as a converging point for multiple biochemical signals, and are involved a large variety of cellular processes such as cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription. Specific antagonists, inhibitors as well as pan-kinase inhibitors can block the interaction signals.
Cell-based kinase signaling pathway LinkLight assays can be utilized to screen and characterize kinase inhibitors. Potentially, non-classic kinase inhibitors could be identified since the assays assess kinase signaling pathways not the phosphorylation status. In addition, the assays could be used to identify small and big molecules that modulate cell-surface receptors via kinase signaling pathways.