Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations.
The simian virus 40 large T antigen is sufficient to confer on cells multiple transformed cell growth characteristics, including growth to a high cell density, rapid growth in medium containing low serum concentrations, and anchorage-independent growth. We showed previously that distinct regions of the protein were involved in conferring these properties and that removal of the first 127 amino acids of T antigen abrogated all three activities. At least three large-T-antigen transformation-related activities have been localized to that region: binding of the tumor suppressor gene product Rb and two independent activities contained within the common region shared by large T and small t antigens. The experiments described here were directed toward determining whether these were the only activities from the N terminus that were needed. To do so we reintroduced an Rb-binding region into the N-terminally truncated T antigen (T128-708) and examined the growth properties of cells immortalized by it in the presence and absence of small t antigen, which can provide the T-common-region transformation-related activities in trans. We show that an Rb-binding region consisting of amino acids 101 to 118, when introduced into a heterologous site in T128-708, is capable of physically binding Rb and that binding is sufficient for cells expressing the protein to acquire the ability to grow to a high saturation density. However, in low-serum medium, the growth rate of the cells and maximal cell density are reduced relative to those of wild-type-T-antigen-expressing cells, and the cells cannot divide without anchorage. This result suggests that although Rb binding is sufficient in the context of T128-708 to confer growth to a high density, one or more other N-terminally located T-antigen activities are needed for cells to acquire the additional growth properties. Small t antigen in trans supplied those activities. These results indicate that the T-common-region activities and Rb binding are the only activities from the T-antigen N terminus needed to restore full transforming activity to the N-terminally truncated T antigen.
Tevethia
M J
MJ
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Lacko
H A
HA
Kierstead
T D
TD
Thompson
D L
DL
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002452:Cell Count; D002455:Cell Division; D002461:Cell Line, Transformed; D003470:Culture Media; D006801:Humans; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D000952:Antigens, Polyomavirus Transforming; D003470:Culture Media; D016160:Retinoblastoma Protein