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Atypon Free PMC Article

Mol Cell Biol. 1992 Nov;12(11):5050-8. doi: 10.1128/mcb.12.11.5050-5058.1992.

Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen.

Molecular and cellular biology

J Dahl, U Thathamangalam, R Freund, T L Benjamin

Affiliations

  1. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.

PMID: 1406680 PMCID: PMC360438 DOI: 10.1128/mcb.12.11.5050-5058.1992
Free PMC Article

Abstract

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.

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