Accumulated helix-destabilizing mutations in the leucine zipper of c-Fos leads to attenuation and temperature sensitivity of function.
Fos protein heterodimerizes through one surface of an alpha-helical domain called the leucine zipper. We have investigated the effect of destabilizing this domain by multiply substituting small residues of its non-interacting surface with glycine. Ternary complex formation between mutated Fos, Jun and DNA was determined in vitro in the presence of denaturant. We also tested the ability of constitutively expressed, mutated Fos proteins to support anchorage independent growth of the cell line Rat1A. Combinations of two substitutions are tolerated in both assays of Fos function, while four substitutions resulted in attenuation in both functions. Rat1A expressing one of the quadruple mutants also showed temperature sensitivity in anchorage independent growth. In dense monolayers of these cells, stromelysin (a Fos-responsive gene product) decreased in abundance as a function of temperature and was less abundant even at 34 degrees C than in cells that overexpressed the wild-type c-fos mRNA. However the mutant transgene itself appeared to show temperature sensitive expression. We suggest that creating a range of glycine substitutions for small residues in the non-interacting face of a leucine zipper might be of general use as a strategy to produce attenuated mutants of other transcription factors.
Forschungs-Institut für Molekulare Pathologie, Wien.
M J
MJ
Nicklin
D
D
Zechner
T
T
Kislyakova
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D015870:Gene Expression; D016762:Genes, fos; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry; D051381:Rats; D013329:Structure-Activity Relationship; D013696:Temperature
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun