Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades.
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- gamma (IFN-gamma)-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-gamma, 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 beta-chain of the IFN-gamma 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.
Stitz
J
J
Department of Microbiology and Immunology, School of Medicine, Stanford University Stanford, CA 94305, USA.
Krutzik
P O
PO
Nolan
G P
GP
D018076:DNA, Complementary / Q000032:analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D000432:Methanol / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017471:Receptors, Interferon / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D020298:U937 Cells
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005404:Fixatives; C086185:IFNGR2 protein, human; D010750:Phosphoproteins; D017471:Receptors, Interferon; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D005557:Formaldehyde; D007371:Interferon-gamma; D000432:Methanol
Methods Mol Biol. 2004;263:67-94
14976361
Nat Immunol. 2003 Nov;4(11):1083-92
14528303
Nature. 2004 Feb 26;427(6977):848-53
14985764
Clin Immunol. 2004 Mar;110(3):206-21
15047199
Cell. 2004 Jul 23;118(2):217-28
15260991
Proc Natl Acad Sci U S A. 1985 Jan;82(1):68-72
3918306
Cell. 1994 Mar 11;76(5):793-802
8124716
Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9146-50
7568090
Immunity. 1998 Apr;8(4):461-71
9586636
J Immunol. 1999 Jan 1;162(1):323-30
9886402
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2129-34
10051606
J Virol. 1999 May;73(5):4470-4
10196349
Nat Genet. 2001 Jan;27(1):23-9
11137994
Nat Biotechnol. 2002 Feb;20(2):155-62
11821861
Immunity. 2002 Jan;16(1):51-65
11825565
Virology. 2001 Dec 20;291(2):191-7
11878888
Nucleic Acids Res. 2002 Aug 15;30(16):e80
12177308
J Biol Chem. 2002 Oct 4;277(40):37512-8
12167667
Cytometry A. 2003 Oct;55(2):61-70
14505311
Pharmacol Ther. 2003 Oct;100(1):1-29
14550502
Science. 2004 Feb 20;303(5661):1192-5
14976315