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-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that 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 carboxy-terminal 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 carboxy-terminal 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 carboxy-terminal 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 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.
Soudant
N
N
Laboratoire d'Oncologie Moléculaire, CNRS URA 1160, Institut Pasteur, Lille, France.
Albagli
O
O
Dhordain
P
P
Flourens
A
A
Stéhelin
D
D
Leprince
D
D
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D007532:Isoleucine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; C494070:Ets2 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015682:Retroviridae Proteins, Oncogenic; D015534:Trans-Activators; D014157:Transcription Factors; C050379:oncogene proteins v-ets; D007532:Isoleucine; D004247:DNA