Nucleic Acids Research, 1994, Vol. 22, No. 19 3871-3879
© 1994
MOLECULAR BIOLOGY |
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
Laboratoire d'Oncologie Moléculaire CNRS URA 1160, Institut Pasteur, 1 Rue Calmette, 59019 Lille Cédex, France
*To whom correspondence should be addressed at: U 124 INSERM, IRCL, 1 Place de Verdun, 59045 Lille Cédex, France
Received July 7, 1994. Accepted August 14, 1994.
The c-ets-1 locus encodes two transcription factors, p54c-ets-1 antj p68c-ets-1 tnat recognize purine-rich motifs. The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68c-ets-1 by two amino acid substitutions (alanine 285 and isoleucine 445 in c-ets-1 both substituted by valine in v-ets, mutations A and B respectively) and its carboxyterminal end (mutation C). The B mutation affects a well conserved residue in the carboxy-terminal 85 amino acids, ETS DNA-binding domain. To address the biological relevance of the B mutation found between v-ets and c-ets-1, we have randomly mutagenized isoleucine 445 of p68c-ets-1 by polymerase chain reaction. Using in vitro gel mobility shift assays, we show that this residue is crucial for the binding properties of c-ets-1 since the 12 mutations we have generated at this position, all diminish or even abolish the binding, to the ‘optimized’ Ets-1 binding site (EBS), of 35 kDa proteins corresponding to the 311 carboxyterminal residues of c-ets-1. Among them, substitutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects. Similar results were obtained when the same mutations were introduced either in full-length p68c-ets-1 protein or into a carboxyterminal polypeptide of 109 amino acids encompassing he 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 p68c-ets-1 that decrease binding activity to EBS abrogate its ability to transactivate reporter plasmids carrying either the TPA Oncogene Response Unit of the Polyoma virus enhancer (TORU) or a sequence derived from the HTLV-1 LTR. Furthermore, as this isoleucine residue is rather well-conserved within the ETS gene family, we show that mutation of the corresponding isoleucine of c-ets-2 into glycine also abrogates its DNA-binding and hence, transactivating properties. Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-1 and c-ets-2 DNA-binding domains.
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