A RESIDUE OF THE ETS DOMAIN MUTATED IN THE V-ETS ONCOGENE IS ESSENTIAL FOR THE DNA-BINDING AND TRANSACTIVATING PROPERTIES OF THE ETS-1 AND ETS-2 PROTEINS

The c-ets-l locus encodes two transcription factors, p54(c-ets-1) and p68(c-ets-1) that recognize purine-rich motifs. The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68(c-et s-1) by two amino acid substitutions (alanine 285 and isoleucine 445 i n c-ets-1 both substituted by valine in v-ets, mutations A and B respe ctively) and its carboxyterminal end (mutation C). The B mutation affe cts a well conserved residue in the carboxy-terminal 85 amino acids, E TS DNA-binding domain. To address the biological relevance of the B mu tation found between v-ets and c-ets-l, we have randomly mutagenized i soleucine 445 of p68(c-ets-1) by polymerase chain reaction. Using in v itro gel mobility shift assays, we show that this residue is crucial f or the binding properties of c-ets-l since the 12 mutations we have ge nerated at this position, all diminish or even abolish the binding, to the 'optimized' Ets-l binding site (EBS), of 35 kDa proteins correspo nding to the 311 carboxyterminal residues of c-ets-l. Among them, subs titutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects. Similar results were obtained when the sam e mutations were introduced either in full-length p68(c-ets-1) protein or into a carboxyterminal polypeptide of 109 amino acids encompassing the ETS-domain which has previously been shown to display a very high binding activity as compared with the full-length protein. Consistent with the in vitro results, point mutations in p68(c-ets-1) that decre ase binding activity to EBS abrogate its ability to transactivate repo rter plasmids carrying either the TPA Oncogene Response Unit of the Po lyoma virus enhancer (TORU) or a sequence derived from the HTLV-l LTR. Furthermore, as this isoleucine residue is rather well-conserved with in the ETS gene family, we show that mutation of the corresponding iso leucine of c-ets-2 into glycine also abrogates its DNA-binding and hen ce, transactivating properties. Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-l and c-ets -2 DNA-binding domains.