SPOT-14 PROTEIN-PROTEIN INTERACTIONS - EVIDENCE FOR BOTH HOMODIMER 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 r apidly regulated by hormones and dietary constituents. We recently sho wed 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 transcrip tion by S14, we employed a yeast two-hybrid system to identify hepatic proteins that physically interact with S14. We found that S14 has a s trong propensity for homodimerization, as is the case for many transcr iption factors. Relevance of this finding to mammalian cells was estab lished by transient cotransfection of S14 constructs bearing two diffe rent 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 co nserved 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 e xtracts of radiolabeled rat hepatocytes. We propose that S14 is an aci dic transcriptional activator that acts as a homodimer to modulate gen e expression as a component of a tripartite complex with a 36-kDa hepa tic protein.