Spot 14 protein-protein interactions: evidence for both homo- and heterodimer formation in vivo.
Spot 14 (S14) is a nuclear protein that is abundant only in lipogenic tissues (liver, adipose, lactating mammary), where its expression is rapidly regulated by hormones and dietary constituents. We recently showed that S14 acts at the transcriptional level in the transduction of signals for increased expression of genes encoding lipogenic enzymes. To better understand the mechanism of the regulation of gene transcription by S14, we employed a yeast two-hybrid system to identify hepatic proteins that physically interact with S14. We found that S14 has a strong propensity for homodimerization, as is the case for many transcription factors. Relevance of this finding to mammalian cells was established by transient cotransfection of S14 constructs bearing two different epitope tags. Glutathione-S-transferase-S14 and hemagglutinin-S14 fusions copurified from the transfected cells by glutathione-affinity chromatography, indicating their association in vivo. Analysis of S14 deletion mutants in the yeast system showed that an evolutionarily conserved hydrophobic heptad repeat (zipper) near the carboxyl terminus was necessary for homodimerization. In parallel studies, we observed a 36-kDa protein that specifically coimmunoprecipitated with S14 from extracts of radiolabeled rat hepatocytes. We propose that S14 is an acidic transcriptional activator that acts as a homodimer to modulate gene expression as a component of a tripartite complex with a 36-kDa hepatic protein.
Cunningham
B A
BA
Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Maloney
M
M
Kinlaw
W B
WB
D000818:Animals; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D017353:Gene Deletion; D015723:Gene Library; D006824:Hybridization, Genetic; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008970:Molecular Weight; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D012440:Saccharomyces / Q000235:genetics; D014157:Transcription Factors
D018076:DNA, Complementary; D009687:Nuclear Proteins; D011506:Proteins; C051065:THRSP protein, human; C501478:Thrsp protein, rat; D014157:Transcription Factors