We constructed a set of plasmids encoding fusion proteins was fused

Notably, EBC5-16 induced a statistically-significant 50% increase in CAT activity compared to TC2-3, indicating that the transmembrane domain of EBC5-16 forms a stronger oligomer than TC2-3. This finding corroborates the biochemical results that a higher fraction of EBC5-16 is present as a dimer in murine cells. The results presented above Echinatin demonstrated that EBC5-16 displays increased dimerization compared to TC2-3. To assess the importance of dimerization in EBC5-16 activity, we mutated both cysteines in the C-terminus of EBC5-16 to serine. This mutant was expressed in BaF3/HAhEPOR cells, and growth factor independence was assessed. As shown in Figure 5C, EBC5-16-CCSS did not confer growth factor independence, demonstrating that the cysteines, and presumably dimerization, are necessary for EBC5-16 activity. Taken together, these results raised the possibility that the increased activity of EBC5-16 is due to increased dimerization. To determine which amino acids constitute the homodimer interface of EBC5-16, we used an approach we developed to identify the dimer interface of the BPV E5 oncoprotein, which was subsequently confirmed by biophysical studies. We constructed a set of plasmids encoding fusion proteins in which EBC5-16 was fused at seven consecutive residues to the dimerization domain of the yeast transcription factor, Put3, Isoliquiritigenin containing an N-terminal AU1 epitope tag. This segment of Put3 contains a leucine zipper motif that forms a left-handed coiled-coil homodimer, which will in essence force the fused protein of interest into a left-handed coiled-coil, whose interface residues can be predicted from the known structure of the Put3 dimer and the point of fusion. By fusing the Put3 segment at sequential residues of EBC5-16, each of the seven possible lefthanded coiled-coil helical registers of the dimeric EBC5-16 segment is generated. The residues that constitute the homodimer interface of native EBC5-16 can be inferred from the fusion protein that displays the highest biological activity. Each of the Put3/EBC5-16 chimeras was cloned into the pRVY-puro vector and used to infect BaF3/HA-hEPOR cells.