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At least 19 recordsLinked to original sources
PubMed2003

Suppression of AP-1 constitutive activity interferes with polyomavirus MT antigen transformation ability.

Polyomavirus (Py) encodes a potent oncogene, the middle T antigen (MT), that induces cell transformation by binding to and activating several cytoplasmic proteins which take part in transduction of growth factors-induced mitogenic signal to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation although, its activation was not sufficient for establishment of the transformed phenotype. Here we show that expression of a dominant-negative cJun mutant in MT transformed cell lines inhibits its transformation ability, indicating that constitutive AP-1 activity is necessary for cell transformation mediated by MT. Evidences also suggest that proliferation of MT transformed cells in low serum concentrations and their ability to form colonies in agarose are controlled by distinct mechanisms.

Animals↗
PubMed1987

Analysis of polyomavirus middle-T-antigen-transformed rat cell variants expressing different levels of pp60c-src.

We characterize two independent variant cellular clones which arose following in vitro passage of polyomavirus middle-T-antigen (MTAg)-transformed FR3T3 cells expressing RNA complementary to c-src mRNA. These clones were initially flat and underwent morphologic transformation at a high frequency to a phenotype indistinguishable from that of parental MTAg-transformed FR3T3 cells. Biochemical analysis of the flat clones prior to phenotypic conversion revealed that these cells synthesized little detectable pp60c-src and had correspondingly low levels of pp60c-src protein kinase activity and MTAg-associated protein kinase activity. The flat cell clones did not possess detectable focus-forming activity, were not capable of detectable anchorage-independent growth, and had saturation densities and doubling times below those normally observed for FR3T3 cells. Following conversion of the flat clones to a shape resembling that of typical MTAg-transformed cells, the abundance of pp60c-src, pp60c-src kinase activity, and MTAg-associated in vitro protein kinase activity were all restored to the levels found in the parental MTAg transformants. These cells had growth rates, focus-forming activities, anchorage-independent growth rates, and saturation densities similar to those of the parental MTAg-transformed rat cells. These data provide additional evidence that maintenance of a transformed phenotype by polyomavirus MTAg in established rat cell lines depends, at least in part, on a minimal threshold level of pp60c-src.

Animals↗
PubMed1986

Significance of the gastrin homology and surrounding sequences in polyomavirus middle T antigen for cell transformation.

Deletion of residues 305 to 327 of polyomavirus middle T antigen, including the (Glu)6-Tyr-315 sequence that is a preferred site of phosphorylation in vitro by pp60c-src, markedly altered viral transformation of rat cells. The efficiency of transformation by the deletion mutant depended on how it was introduced into cells, and the resulting transformants displayed limited growth rates in monolayer and in suspension. Substitution of the polyomavirus residues 305 to 327 with a homologous region (containing [Glu]5-Ala-Tyr) from porcine gastrin did not restore wild-type transforming activity. These mutant middle T antigens interacted with pp60c-src and were phosphorylated in vitro. Thus, although a sequence of consecutive glutamic acid residues followed by a tyrosine is a dominant structural element which strongly influences the physical properties of middle T antigen, its presence did not ensure the biological activity of the protein. Other elements in this region of middle T antigen also contributed substantially to the transforming capacity of polyomavirus.

Amino Acid Sequence↗
PubMed1991

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.

Animals↗
PubMed1990

The amino terminus of polyomavirus middle T antigen is required for transformation.

In polyomavirus-transformed cells, pp60c-src is activated by association with polyomavirus middle T antigen. These complexes have a higher tyrosine kinase activity compared with that of unassociated pp60c-src. Genetic analyses have revealed that the carboxy-terminal 15 amino acids of pp60c-src and the amino-terminal half of middle T antigen are required for this association and consequent activation of the tyrosine kinase. To define in greater detail the borders of the domain in middle T antigen required for activation of pp60c-src, we constructed a set of unidirectional amino-terminal deletion mutants of middle T antigen. Analysis of these mutants revealed that the first six amino acids of middle T antigen are required for it to activate the kinase activity of pp60c-src and to transform Rat-1 fibroblasts. Analysis of a series of insertion and substitution mutants confirmed these observations and further revealed that mutations affecting the first four amino acids of middle T antigen reduced or abolished its capacity to activate the kinase activity of pp60c-src and to transform Rat-1 cells in culture. Our results suggest that the first four amino acids of middle T antigen constitute part of a domain required for activation of the pp60c-src tyrosyl kinase activity and for consequent cellular transformation.

Amino Acid Sequence↗
PubMed1992

Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation.

Polyomavirus middle T antigen mediates transformation of cells, at least in part, by its association with and activation of the intrinsic protein tyrosine kinase activity of pp60c-src. pp60c-src, by analogy with pp60v-src, elicits cell proliferation through a signal transduction pathway that includes p21c-ras. Therefore, we tested the possibility that middle T antigen acts upstream of and in the same proliferative signaling pathway as p21c-ras. Co-transfection of Rat-2 cells with plasmids expressing human Krev-1, a dominant suppressor of Ki-ras transformation, and mT antigen resulted in a dose-dependent reduction of mT antigen-induced foci. Krev-1 did not affect the transforming activity of SV40 large T antigen, demonstrating that the transformation-suppressing activity of Krev-1 is specific. To determine the effect of Krev-1 on stably transformed cell lines, Krev-1 DNA was introduced into middle T antigen-transformed Rat-2 cells along with a G418 resistance marker. Of the G418-resistant colonies examined, 1% were morphologically untransformed. Characterization of several morphological revertants revealed that, with the exception of one cell line, all of the cell lines expressed middle T antigen, which was associated with pp60c-src, whose tyrosine kinase activity was similar to that found in the parental transformed cell lines. To determine whether other phenotypic traits associated with transformation were altered in these cell lines, their growth rates and ability to form colonies in agar suspension were examined. The majority of the revertants had longer doubling times, and grew less efficiently in agar suspension compared with their transformed parents. A direct correlation was observed between Krev-1 RNA and protein expression and the efficiency with which the revertants formed colonies in suspension. These results suggest that p21c-ras lies downstream of middle T antigen and pp60c-src in the same proliferative signal transduction pathway.

Animals↗
PubMed1987

Cooperation of middle and small T antigens of polyomavirus in transformation of established fibroblast and epithelial-like cell lines.

We have reported recently that small T antigen of polyomavirus stimulates the growth of NIH 3T3 cells beyond their saturation density and induces weak anchorage-independent growth (T. Noda, M. Satake, T. Robins, and Y. Ito, J. Virol. 60:105-113, 1986). We examined whether small T antigen would cooperate with middle T antigen in the in vitro transformation of NIH 3T3 (fibroblasts) and NRK-52E (epitheliallike) cells. The small-T-antigen gene, when cotransfected with the middle-T-antigen gene, had no additional effect on the efficiency or size of dense foci formation induced by the middle-T-antigen gene on a monolayer of NIH 3T3 cells. However, the small-T-antigen gene dramatically increased the rate of growth of NIH 3T3 cells transformed by middle T antigen in semisolid medium. Introduction of the small-T-antigen gene into middle-T-antigen-transformed cells did not disturb the integrated middle-T gene, alter expression of the middle-T gene, or enhance middle-T-antigen-associated tyrosine protein kinase activity. For NRK-52E cells, the expression of middle T antigen alone resulted in small, slow-growing foci on a monolayer. These cells did not show anchorage-independent growth, despite the fact that middle-T-antigen-associated tyrosine protein kinase activity was clearly detected in these cells. NRK-52E cells expressing both middle and small T antigens formed faster growing foci on a monolayer than middle-T-antigen-expressing cells did and grew in semisolid medium, even when the amounts of middle T antigen and its associated kinase activities were lower than those of middle-T-antigen-expressing cells. We conclude that small T antigen cooperates with middle T antigen in the in vitro transformation of established cell lines of fibroblast and epitheliallike cells, that it does not share the middle-T-antigen function even though they are structurally related, and that it has a significantly more important role in the transformation of NRK-52E cells than that of NIH 3T3 cells.

Animals↗
PubMed1986

Mapping of phosphorylation sites in polyomavirus large T antigen.

The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

Amino Acids↗
PubMed1986

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.

Amino Acids↗
PubMed1986

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

Animals↗
PubMed1984

The major site of tyrosine phosphorylation in polyomavirus middle T antigen is not required for transformation.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.

Amino Acid Sequence↗
PubMed1985

Selection of transformed cells in serum-free media.

NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated human ras gene, and the mouse c-myc gene.

Animals↗
PubMed1994

Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases.

BACKGROUND: Expression of polyomavirus middle T antigen (PymT) rapidly induces endothelial tumors (hemangiomas) in mice, with an apparent single rate-limiting step. Because activation of Src-like kinases is thought to be an important component of PymT-induced transformation, we have analyzed the functional requirement for individual kinases in this process. This type of analysis has only recently become possible, with the generation of 'gene knock-out' mice lacking each of the kinase genes src, fyn and yes. RESULTS: Hemangiomas develop efficiently in newborn mice lacking either src, fyn or yes after inoculation with a PymT-transducing retrovirus. In src- and fyn-deficient mice, the kinetics of induction and the histological properties of the tumors were indistinguishable from those in wild-type mice. In contrast, a reduced number of tumors arose in yes-deficient mice, with a significantly longer latency period. Transformed endothelial cell lines derived from the induced hemangiomas, however, did not differ in their morphological and tumorigenic properties from cell lines established previously from wild-type mice. Biochemical analysis of complexes between PymT and the Src-related kinases in these cell lines suggests that the Yes kinase is responsible for a significant amount of the PymT-associated kinase activity in transformed endothelial cells. CONCLUSION: We have demonstrated that inactivation of a single tyrosine kinase of the Src family in endothelial cells does not abrogate PymT-induced hemangioma formation. As the remaining kinases do not compensate for the absence of a family member by elevated kinase activity, the loss--which affects the transformation process to varying degrees--can be studied in this model system. Our studies suggest that the PymT-Yes kinase complex plays a major role in the tumor-initiating action of PymT.

Animals↗
PubMed1987

Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein.

Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.

Animals↗
PubMed1986

Properties of cells transformed by the middle T-antigen-coding region of polyomavirus.

A series of 10 Fischer rat transformed clonal cell lines were independently obtained in infections with a defective polyomavirus containing a scrambled genome except for an intact middle and small T-antigen-coding region. These cells synthesize middle and small T antigens; no fragment of large T antigen can be detected in any of them. The transformed phenotype of this set of cell lines (designated LT-) has been studied with respect to serum dependence, saturation density, and anchorage independence and compared with the phenotype of a set of six transformants (designated LT+) which synthesize detectable to high levels of shortened or normal-sized large T antigen. Both the LT+ and the LT- groups of polyomavirus transformants display a range of transformed phenotypes. These ranges overlap, and the variations within each group are larger than the variations between the two groups. Thus, the results suggest that, for established Fischer rat fibroblasts, the maintenance of any of the three phenotypes tested and, in particular, of serum independence is not necessarily correlated with the levels of large T antigen or fragments thereof.

Animals↗
PubMed1985

Recent progress in studies of polyomavirus tumour antigens.

The polyomavirus tumour (T) antigens were originally identified by their reactivity with antisera from tumour-bearing animals. The primary structure of the three T-antigens has been established by combining the information from the nucleotide sequencing of DNA, RNA analysis, and peptide mapping. The functions of the T-antigens in productive infection and cellular transformation have largely been analysed by using virus mutants. The large T-antigen binds specifically to polyomavirus DNA. This binding is probably linked to the activity of the protein in the control of viral DNA and RNA synthesis. In addition, the large T-antigen has the ability to confer an unlimited growth potential to cells in culture. The middle T-antigen is a primary inducer of cellular transformation. The part of this protein that is located in the plasma membrane, is associated with a tyrosine kinase activity. The small T-antigen, finally, has not yet been studied extensively. However, small T-antigen has to be expressed to allow a complete productive infection cycle in mouse cells.

Amino Acids↗
PubMed1988

A polyomavirus tumor-specific transplantation antigen (TSTA) epitope is situated within the N-terminal amino acid sequence common to middle and small T-antigens.

An MT cDNA-transformed rat cell line (2.8), that contains only the polyoma middle T-antigen and expresses polyoma TSTA, can be rejected in rats immunized with 1837 cells that carry a host range (A185 hr-t), mutant which expresses a full-length large T-antigen, and nonfunctional N-terminal fragments of small and middle T. This shows that at least one polyoma TSTA epitope is situated in the 113 amino acid N-terminal region, which is common to middle and small T-antigens.

Amino Acid Sequence↗
PubMed1986

SV40 large T-antigen: dual oncogene.

Simian virus 40 (SV40) is a small, DNA-containing tumour virus. One of its gene products, the large tumour antigen (T-ag), is essential for both viral replication and cell transformation. SV40 T-ag can be considered a dual oncogene protein; it is a composite transforming protein that provides distinct functions at different subcellular locations. In addition to its roles in virus replication, T-ag exerts numerous effects on host cells. Those cellular effects reflect viral stimulation of host cell entry into S phase. Numerous chemical modifications have been ascribed to T-ag. They might be involved in defining subpopulations of T-ag that are, in turn, responsible for mediating various T-ag biochemical functions. The T-ag polypeptide, 90,000-100,000 in molecular weight, appears to contain multiple, discrete functional domains; several biological activities have been assigned to relatively small defined regions of the molecule. The cellular progenitors of the T-ag biochemical activities are not obvious. A cellular protein, p53, thought to be involved in regulation of cell proliferation, becomes complexed with T-ag in transformed cells and is stabilized. The interaction of T-ag with this cellular substrate may play an important part in SV40 transformation. T-ag and T-ag/p53 complexes are localized in both the nucleus and plasma membrane of transformed cells. T-ag is transported to the nucleus because of a 7-residue nuclear transport signal contained within its primary sequence. Its migration to the membrane is by an unknown pathway. Only a minor fraction of the total cellular T-ag is present at the cell surface. Both amino and carboxy termini of the T-ag polypeptide are exposed on the extracellular face of the cell. Nuclear and membrane T-ag are structurally very similar, although a portion of membrane T-ag is acylated and nuclear T-ag is not. The nuclear and membrane forms of T-ag apparently provide separate and complementary functions necessary for cell transformation. Nuclear T-ag is important in immortalizing primary cells and membrane T-ag may mediate more pronounced morphological changes. A model is presented, postulating how the two forms of T-ag might cooperate to mediate phenotypic transformation.

Animals↗