Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades

Abstract

We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon γ (IFN-γ)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-γ, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the β-chain of the IFN-γ receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen. © The Author 2005. Published by Oxford University Press. All rights reserved.