Oncogenic transformation by the FOX protein Qin requires DNA binding.
Some functions of the Qin oncoprotein are not dependent on DNA binding. In order to test the requirement for DNA binding in Qin-induced oncogenic transformation, site directed mutations were introduced in the winged helix (WH) DNA binding domain of the Qin protein. In cellular Qin (c-Qin), the glycine at position 233 was either deleted or substituted with the amino acids aspartic acid, alanine, glutamic acid, asparagine, proline or lysine. The same position carries aspartic acid in the viral Qin protein (v-Qin). The adjacent residues, threonine 232 and lysine 234, were separately mutated to proline. Several additional amino acid substitutions believed to be involved in DNA contacts were introduced at the following c-Qin positions: asparagine 189, histidine 193, serine 196 or arginine 236. Most of the substitutions reduced DNA binding of Qin, one mutation, H193A, completely abolished DNA binding, and another mutation, T232P, increased DNA binding affinity. Mutant H193A failed to transform chicken embryo fibroblasts (CEF), all other mutants, even those showing minimal DNA binding, retained oncogenicity for CEF. The efficiencies of focus formation induced by these mutant proteins in cell culture were not significantly different from that of wild type. However, the rate of focus development and the size of foci induced by the Qin mutants were greater with strong DNA binders than with weak DNA binders. Transdominant negative constructs consisting of the winged helix domain of cQin or v-Qin interfered with focus formation induced by full length Qin proteins. These results suggest that DNA binding is a prerequisite for transformation by Qin, and strong DNA binding is related to accelerated transformation in CEF.
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Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Geerdes
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Vogt
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PK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D030161:Avian Proteins; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001665:Binding Sites; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002472:Cell Transformation, Viral / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000378:metabolism*; D005347:Fibroblasts; D051858:Forkhead Transcription Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D030161:Avian Proteins; D051858:Forkhead Transcription Factors; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C415088:oncogene protein Qin, Avian retrovirus; C490425:v-qin protein, Avian sarcoma virus 31; D004247:DNA