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Genetic analysis of signaling pathways in human cells
Author(s) -
Stark George R.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.19.1
Subject(s) - biology , gene , transcription factor , genetics , mutagenesis , promoter , insertional mutagenesis , mutant , microbiology and biotechnology , gene expression
In early work, selection of cells resistant to N‐phosphonacetyl‐L‐aspartate (PALA), a potent inhibitor of aspartate transcarbamylase, helped to reveal gene amplification as a mechanism of drug resistance. Later, to learn how cells respond to interferons, we treated them with a frameshift mutagen, isolating clones lacking expression of eight different proteins required for signaling, thus helping to identify and characterize the JAK‐STAT pathways of response to many cytokines. More recently, we used an efficient method for inserting a strong promoter randomly into the genomes of cells to up‐regulate genes responsible for drug resistance, transformation and signaling, facilitating the new discovery that the inducible transcription factors NFκB and STAT3 are modified by reversible lysine methylation. The responsible methyl transferases and demethylases also modify histones, and the methylations of NFκB and STAT3 occur when these proteins are bound to specific promoters. Methylations of K218 and K221 of the p65 subunit of NFκB are essential for activity, probably because they strengthen binding to DNA. Additional discoveries made by using insertional mutagenesis will be discussed.