Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen.
The functional importance of the two clusters of positively charged amino acids which flank the hydrophobic membrane-anchoring sequence of polyomavirus middle T (mT) protein has been investigated by using site-directed mutagenesis. A clear asymmetry was apparent. No effect on transformation was seen following multiple alterations or complete removal of the cluster at the carboxyl end of the protein. In contrast, a single substitution replacing the first arginine amino terminal to the hydrophobic stretch with glutamic acid, but not with lysine, histidine, or methionine, produced a partially transformation-defective mutant with a novel phenotype. This mutant failed to confer anchorage-independent growth on F111 established rat embryo fibroblasts but induced foci with altered morphology compared with wild-type mT. Biochemical studies on this mutant revealed that F111 clones expressing levels of mutant mT equivalent to those of wild-type controls showed a 65% reduction in pp60c-src activation and an 87% reduction in mT-associated phosphatidylinositol 3-kinase activity. However, F111 clones expressing seven times more mutant mT than did wild-type controls showed equal or greater levels of kinase activities yet remained incompletely transformed. Possible mechanisms involving this transformation-sensitive region of mT are discussed.
Dahl
J
J
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
Thathamangalam
U
U
Freund
R
R
Benjamin
T L
TL
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000382:microbiology*; D002472:Cell Transformation, Viral / Q000502:physiology; D004247:DNA; D004594:Electrophysiology; D005347:Fibroblasts / Q000166:cytology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats
D000952:Antigens, Polyomavirus Transforming; D004247:DNA