Biomedical subjects

W Markland

Publications and source records attributed to W Markland.

23 records · Page 2Linked to original sources

PubMed1987

Mapping of the amino-terminal half of polyomavirus middle-T antigen indicates that this region is the binding domain for pp60c-src.

The majority of the carboxy-terminal half of polyomavirus middle-T antigen has been variously mutated and, with the exception of the putative membrane-binding domain (amino acids 394 to 415), was found to be largely dispensible for the transforming activity of the protein. A comparison of the small-T antigen amino acid sequences (equivalent to the region of middle-T encoded by exon 1) of simian virus 40, BK virus, polyomavirus, and a recently described hamster papovavirus highlighted regions of potential interest in mapping functions to the amino-terminal half of polyomavirus middle-T antigen. The regions of interest include amino acids 168 to 191 (previously investigated by this group [S. H. Cheng, W. Markland, A. F. Markham, and A. E. Smith, EMBO J. 5:325-334, 1986]), two cysteine-rich clusters (amino acids 120 to 125 and 148 to 153), and amino acids 92 to 117 (within the limits of the previously described hr-t mutant, SD15). Point mutations, multiple point mutations, and deletions were made by site-specific and site-directed mutagenesis within the cysteine-rich clusters and residues 92 to 117. Studies of the transforming ability of the altered middle-T species demonstrated that this activity is highly sensitive to amino acid changes. All four regions (as defined above) within the amino-terminal half of middle-T have now been studied in detail. The phenotype of the mutants is predominantly transformation defective, and the corresponding variant middle-T species are characterized by being either totally or severely handicapped in the ability to associate actively with pp60c-src. Whether the mutations affect the regions of interaction between middle-T and pp60c-src or simply interfere with the overall conformation of this domain is not known. However, there would appear to be a conformational constraint on this portion of the molecule with regard to its interaction with pp60c-src and by extension to the ability of the middle-T species to transform.

Amino Acid Sequence↗

PubMed1987

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.

Animals↗

PubMed1986

Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation.

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.

Amino Acid Sequence↗

PubMed1986

In vitro mutagenesis of the putative membrane-binding domain of polyomavirus middle-T antigen.

Polyomavirus middle-T antigen contains a contiguous sequence of 22 hydrophobic amino acids near the carboxyl terminus, which is the putative membrane-binding domain of the protein. The DNA encoding this region was mutated to form a series of deletions, insertions, and substitutions called RX mutants. The phenotypes of these mutants fall into three groups based on the transforming and biochemical properties of their encoded proteins. The first group, with deletions outside but proximal to the hydrophobic domain, displayed an essentially wild-type phenotype. A second group, with extensive deletions within the region encoding the hydrophobic domain, expressed middle-T species which did not fractionate with cellular membranes or associate with pp60c-src and which were defective in their ability to transform. A third group of mutants with more subtle predicted alterations in the hydrophobic domain were wild type for the biochemical parameters investigated but were unable to transform cultured rodent cells. These observations are consistent with previous findings that membrane association plays an important role in transformation by middle-T and that, whereas association between middle-T and pp60c-src is a necessary correlate of transformation, it is not sufficient. A comparison of murine polyomavirus middle-T and a newly described hamster papovavirus putative middle-T revealed a strong homology between their respective hydrophobic-domain amino acid sequences. This homology is not observed in the anchorage domains of other model proteins, and this may imply that the middle-T hydrophobic domain is important in transformation for reasons other than simple membrane association.

Animals↗

PubMed1986

Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250.

Tyrosine residues of middle-T and tyrosine phosphorylation are thought to be important in the transformation of cultured rodent cells by polyomavirus. Of the potential tyrosine sites in the carboxyl-terminal half of middle-T, tyrosines 297, 315, and 322 have been studied previously, whereas tyrosine 250 has not. Two mutant plasmids, XD121 and pT250, encode polyomavirus middle-T species in which the tyrosine 250 residue is affected. XD121 is a deletion mutant in which the region encoding tyrosine 250, together with three adjacent amino acids, is deleted, whereas pT250 is a point mutant in which the tyrosine 250 codon has been converted to a phenylalanine codon. The plasmids were handicapped in transforming ability, as judged by focus formation on a monolayer of Rat-1 cells. Both demonstrated a reduction in the number of foci produced and a lag in the time of appearance of foci when compared with wild-type plasmid. The importance of residue 250 in this phenotype was indicated by the observation that plasmids containing multiple mutations proximal to the tyrosine 250 codon were wild type in their transforming ability. Furthermore, a revertant of pT250 (pT250-w.t.), which utilized the alternative tyrosine codon of TAC, was shown to regain full transforming activity. A combined-mutant plasmid, pTH, encodes a middle-T species in which both tyrosines 250 and 315 are converted to phenylalanine. This plasmid was totally defective in the transformation of rodent cells in a focus formation assay; however, it did impart a small measure of anchorage-independent growth when the encoded protein was expressed in NIH 3T3 cells. The in vitro kinase activity and pp60c-src association of the mutant middle-T antigens were examined. These assays demonstrated a reduction in phosphate acceptor activity for the middle-T species encoded by pT250 and pTH. Quantitative kinase assays showed that all of the tyrosine-mutant middle-T species, encoded by pAS131 (containing the tyrosine 315 codon-to-phenylalanine codon mutation), pT250, and pTH, were able to enhance pp60c-src kinase activity but only at levels which were intermediate and which reflected their transforming abilities relative to wild type.

Amino Acid Sequence↗