Application Note | March 27, 2014

A Homogeneous Assay To Quantify Endogenous AKT Phosphorylation In Human Umbilical Vein Endothelial Cells

Source: BioTek Instruments, Inc.

AKT is a serine/threonine protein kinase that plays a key role in cellular processes. AKT has become a popular target for drug discovery campaigns, due to the fact that AKT inhibitors may help to treat a number of cancers. Here we demonstrate an automated homogeneous assay to probe AKT phosphorylation at its serine 473 residue using endogenous levels of kinase expression within human primary HUVEC cells.

The serine/threonine kinase AKT, also known as protein kinase B (PKB), mediates cell survival, therefore inhibiting apoptosis, or cellular death. It also regulates many cellular functions such as cell proliferation and differentiation, cellular migration, glucose and intermediary metabolism, and transcription. Additionally, AKT induces protein synthesis pathways, and is therefore a key signaling protein in the cellular pathways that lead to skeletal muscle hypertrophy, tumor development and general tissue growth. Its implication for cancer research and that of other disease states makes AKT an important target for drug discovery campaigns.

AKT is a key downstream intracellular convergence point for many cell signaling pathways. These diverse signaling pathways are activated by growth factors including vascular endothelial growth factors (VEGFs), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1). AKT phosphorylation is linked through PI-3 kinase activity. AKT possesses a PH binding domain for binding PIP3, the product of PI-3 kinase phosphorylation of PIP2. By binding PIP3, AKT becomes correctively positioned at the membrane for phosphorylation by its activating kinases. One of these, the mammalian target of rapamycin complex 2 (mTORC2), phosphorylates AKT at its serine 473 residue as a first step to AKT phosphorylation by PDK1. Activated AKT can then go on to activate or deactivate its myriad substrates via its own kinase activity.

For full access to this content, please Register or Sign In.

Access Content A Homogeneous Assay To Quantify Endogenous AKT Phosphorylation In Human Umbilical Vein Endothelial Cells