A method for screening substances for effect on cAMP levels based on intracellular translocation of PKA

摘要

Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP,EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappB, CDK2, Grk5, Zap70. p85, protein-tyrosine phosphatase 1C, stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signaling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of influence. Measurement of redistribution is performed by recording of light intensity, fluorescense lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a fluorescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.