Two domains within the adenovirus type 12 E1A unique spacer have disparate effects on the interaction of E1A with P105-Rb and the transformation of primary mouse cells.
Transformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the unique spacer region of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of the spacer binds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a second spacer mutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1 diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awt or, least efficient, E1ASpm2. These properties of the spacer mutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1 (plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2 or Ad12 E1AdelCR2 (lacking the entire CR2) died within 6-10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes approximately 120 h. Moreover, in the established cell lines, the affinity of E1ASpm1 to p105-Rb was higher than with that of E1Awt. Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-unique spacer, whereas the hydrophobic stretch of six alanines in the spacer is required for stable transformation.
Rumpf
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Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Hufelandstrasse 55, Essen, 45122, Germany.
Esche
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Kirch
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D017500:Adenovirus E1A Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002472:Cell Transformation, Viral; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D017500:Adenovirus E1A Proteins; D016160:Retinoblastoma Protein