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Homogeneous Cell-based Signal Transduction AssaysDownload
April 21, 2010
Authors: Brad Larson, Peter Banks, Paul Held; BioTek Instruments, Inc., Winooski, Vermont, USA
Mammalian Target of Rapamycin (mTOR) is a protein kinase predominantly found in the cytoplasm of the cell. It acts as a central regulator of many biological processes that are essential for cell proliferation, angiogenesis, and cell metabolism . mTOR exerts its effects primarily by turning on and off the cell’s translational machinery, resulting in protein synthesis.
mTOR is a key downstream intracellular point of convergence for a number of cellular signaling pathways. These diverse signaling pathways are activated by a variety of growth factors (including vascular endothelial growth factors (VEGFs), plateletderived growth factor (PDGF), epidermal growth factor (EGF), insulinlike growth factor 1 (IGF1)), hormones (insulin, estrogen, progesterone), and the presence or absence of nutrients (glucose, amino acids) or oxygen [4,5]. One or more of these signaling pathways may be abnormally activated in patients with many different types of cancer, resulting in deregulated cell proliferation, tumor angiogenesis, and abnormal cell metabolism [1,4,5].
mTOR exists in two complexes, mTORC1 and mTORC2, both of which contribute to tumor cell growth. Here we provide results for an AlphaScreen assay for measuring mTORC1 phosphorylation of p70 S6 kinase at its threonine(389) residue, both expressed endogenously in the cancer cell line MCF7. mTORC1 activation is produced through insulin stimulation and inhibition with rapamycin demonstrated.
We have developed the assay using standard 2D cell culture techniques as well as the Synergy H4 Hybrid MultiMode Microplate Reader. The tungsten halogen lamp used in the reader represents a simple, yet robust solution to create the high excitation energy necessary for AlphaScreen assays. In addition, the tungsten lamp is a broad band source useful for a wide wavelength range of excitation and can also be used with other detection modes including fl uorescence intensity and fl uorescence polarization assays. Optimization and validation experiments demonstrate how the combination of assay and instrument can provide an easy to use method to examine the function of this important signal transduction pathway.