RESUMO
Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.
Assuntos
Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Polyomavirus/imunologia , Polyomavirus/metabolismo , Proteínas/metabolismo , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Células 3T3 , Animais , Antígenos Transformantes de Poliomavirus/genética , Baculoviridae/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Transformação Celular Neoplásica , Primers do DNA/genética , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Polyomavirus/genética , Infecções por Polyomavirus/etiologia , Ligação Proteica , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Serina/química , Spodoptera , Transfecção , Infecções Tumorais por Vírus/etiologiaRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Membrana Celular/microbiologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Sequência de Bases , Linhagem Celular , Transformação Celular Viral/fisiologia , DNA , Eletrofisiologia , Fibroblastos/citologia , Dados de Sequência Molecular , Mutagênese , Mapeamento de Peptídeos , Ligação Proteica , RatosRESUMO
In most murine cells transformed by the SV40 virus, virtually all of the cellular phosphoprotein p53 is in a complex with the SV40 T antigen. Here, we report that, in SV40-infected T-antigen-positive Balb 3T12 mouse cells, most (approximately 80%) of the p53 is not in complex. Complex formation was determined by measuring the amounts of [35S]methionine-labeled p53 which coprecipitated with T antigen when using monoclonal antibody to T antigen. The amount of complex formation was expressed as a percentage of total p53 present, measured by the amount of p53 precipitated with the monoclonal antibody to the p53. The values were confirmed by Western blotting procedure, in which the steady-state levels of the proteins were measured. In these measurements after complete precipitation with antibody to T antigen, the residual p53 in the supernatant was precipitated by antibody to p53, and this amount was denoted as free p53. There was no significant difference seen between the [35S]methionine-labeled tryptic peptides of complexed and the free p53 (or between complexed and free T antigens) as determined by two-dimensional gel electrophoresis and chromatography. Virus rescue experiments and retransformation by the rescued virus showed that there was no mutation in the SV40 DNA coding for the T antigen which could account for the lack of complex formation. Both p53 and T antigen were underphosphorylated in cells which exhibited reduced complex formation. Tumorigenicity in syngeneic mice and anchorage-independent cell growth in culture of various cloned mouse cells with or without T antigen expression was compared. The changes in the biologic properties were explainable solely on the basis of known or expected effects of expression of the T antigen and were independent of complex formation or of absence of complex formation between p53 and T antigen.
