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W Deppert

Publications and source records attributed to W Deppert.

At least 91 records · Page 5Linked to original sources
PubMed1990

Functional interaction of nuclear transport-defective simian virus 40 large T antigen with chromatin and nuclear matrix.

We analyzed the subcellular distribution of nuclear transport-defective simian virus 40 Lys-128-mutant (cT-3 [R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984] and d10 [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith, Nature (London) 311:33-38, 1984]) large T antigens in various Lys-128-mutant-transformed rodent cells and in Lys-128-mutant d10-infected TC7 cells. Small but significant amounts of the mutant large T antigens were found in association with nuclear substructures, both in mutant-transformed and in mutant-infected cells. Experiments with TC7 cells made incompetent for cell division by 60Co irradiation supported the assumption that Lys-128-mutant large T antigen did not associate with nuclear components during mitosis but most likely was transported into the nucleus because the Lys-128 mutation was leaky for nuclear transport. Low-level simian virus 40 DNA replication and production of infectious mutant virus progeny in TC7 cells indicated that the association of Lys-128-mutant large T antigen with nuclear substructures is functional.

Animals↗
PubMed1990

Phosphorylation of p53 in primary, immortalised and transformed Balb/c mouse cells.

To address the question whether phosphorylation of p53 might be functionally involved in its metabolic stabilisation and in cellular transformation processes, we have analysed the phosphorylation of the cellular protein p53 in normal and transformed cells of Balb/c mouse origin. Two-dimensional tryptic peptide maps of metabolically unstable p53 from normal Balb/c 3T3 cells and of metabolically stable p53 from 3T3 cells transformed by Simian virus 40 (SV3T3 cells) revealed no qualitative differences between their phosphorylation sites. Except for the unique lack of one or possibly two sites, the phosphopeptide map of p53 from cells transformed by the chemical carcinogen methylcholanthrene (MethA cells), expressing two different, metabolically stable mutant forms of p53, was identical to that of wild-type p53 from normal and SV40-transformed 3T3 cells. These results suggest that no direct relationship exists between phosphorylation of p53, its metabolic stabilisation, and cellular transformation processes. We have included in our analyses p53 from primary Balb/c mouse embryo fibroblast (MEF) cells and the immortalised MEFP27 cell line, established from primary cells after 27 passages. The phosphorylation sites of p53 from these cells were found to be identical to that of p53 from 3T3 cells. In addition, primary and established (immortalised) Balb/c mouse cells revealed no major differences in abundance of p53 mRNA and p53 protein, as well as stability of p53. These results indicate that immortalisation of normal cells involves neither gross alterations of p53 expression nor changes in specific phosphorylation of p53.

Animals↗
PubMed1990

Cell cycle control by p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. I. Regulation of p53 expression.

In addition to controlling the transition of resting normal cells from the G0-state of the cell cycle into S-phase, expression of the cellular protein p53 also seems to be necessary for the proliferation of cycling normal cells in an as yet undefined manner. To further elaborate the role of p53 in growing cells, we analysed p53 expression and its regulation in cells going into, and after release from, growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, induced by isoleucine depletion. Since growth arrest at the R-point is subject to internal control mechanisms of the cell cycle, this approach allowed us to include in our analyses normal Balb/c 3T3 fibroblasts, as well as cells of the chemically induced Balb/c fibrosarcoma cell line Meth A, expressing mutated p53. Isoleucine depletion induced a viable growth arrest at the R-point in cells of both cell lines, marked by a synchronous shut-down of DNA synthesis when the cells went into growth arrest, and a synchronous resumption of DNA synthesis after a lag period of about 2-4 h when the cells were released from growth arrest, as well as a shift to a G1 DNA content at the R-point. p53 expression in both cell lines showed a phenotypically similar regulation, as its synthesis was specifically reduced at the R-point. At the molecular level, however, p53 expression in growth arrested 3T3 cells was controlled at the transcriptional/post-transcriptional level, whereas control in growth arrested Meth A cells seemed to be at the level of mRNA translation. After release from growth arrest, p53 synthesis in both types of cells was rapidly restored, preceding resumption of total protein synthesis, and exhibiting a p53-specific profile.

Animals↗
PubMed1990

Cell cycle control of p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. II. Requirement for cell cycle progression.

To further characterize the role of p53 in growing normal Balb/c 3T3 fibroblasts, as well as of p53 in cells of the methylcholanthrene induced fibrosarcoma cell line Meth A, we analysed the effect of inhibition of p53 synthesis by microinjection of p53-specific monoclonal antibody PAb 122 into the nuclei of these cells after release from growth arrest induced by isoleucine starvation (see preceding paper [Steinmeyer et al., this issue] ). We show that microinjection of PAb 122, but not of control immunoglobulins, into the nuclei of both types of cells effectively blocked their re-entry into the S-phase of the cell cycle. Since isoleucine depletion of these cells was shown to lead to a growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, our results (i) define more precisely the role of p53 in growing cells as a protein controlling transition of the cells through this restriction point, and (ii) demonstrate that mutated p53 in Meth A cells still is functional with regard to cell cycle control at this restriction point. We suggest that p53 acts as a 'gate-keeping' protein at restriction points in the cell cycle, exerting a positive effect on the transition of cells through the cell cycle.

Animals↗
PubMed1989

Quantitative analysis of cell surface-associated SV40 large T antigen using a newly developed 3H-protein A binding assay.

We have established a sensitive assay for the quantitative determination of large T antigen determinants on the surface of living simian virus 40 (SV40)-transformed cells (mKSA). Cells in suspension culture were incubated with monoclonal antibodies specific for large T antigen (KT3, directed against the carboxyterminus of large T antigen, and PAb 108, directed against an aminoterminal determinant on large T antigen). After incubation with secondary antibody (rabbit anti-mouse IgG), followed by incubation with 3H-protein A, the cells were sequentially extracted first with the nonionic detergent NP-40, followed by ultrasonication and extraction with the zwitterionic detergent Empigen BB. NP-40 solubilized large T antigen associated with NP-40-soluble constituents of the plasma membrane, whereas Empigen BB solubilized the plasma membrane lamina-associated subclass of large T antigen (U. Klockmann and W. Deppert, 1983, EMBO J., 7, 1151-1157). The amount of cell surface-bound 3H-protein A in the NP-40 and Empigen BB extracts was determined by liquid scintillation counting. In agreement with earlier reports, cell surface large T antigen was mainly found in association with the plasma membrane lamina (PML). Since the specific activity of 3H-protein A was known, it was possible to calculate the number of surface-bound 3H-protein A molecules, and thus to estimate the average number of surface-exposed amino- and carboxyterminal determinants of large T antigen per cell. KT3 recognized about 450-900 carboxyterminal determinants, while PAb 108 bound to about 1200-2400 aminoterminal determinants on the surface of a single mKSA cell. The cellular protein p53 also was detected on the surface of mKSA cells and was found to be present in amounts comparable to cell surface large T antigen.

Animals↗
PubMed1989

Nuclear subcompartmentalization of simian virus 40 large T antigen: evidence for in vivo regulation of biochemical activities.

Simian virus 40 large T antigen (large T) in the early and the late phases of infection differs significantly in its sequence-specific DNA-binding and ATPase activities, indicating that different large-T populations participate in virus-specific events at various stages of the infectious cycle. To further characterize these large-T populations, we have analyzed nuclear subclasses of large T, isolated from their in vivo location, for their biochemical activities. We show that chromatin- and nuclear matrix-associated large-T molecules exhibit different simian virus 40 control region (ORI) DNA-binding and ATPase activities. The association of large T with a certain nuclear substructure, therefore, subcompartmentalizes large-T molecules exerting different biochemical activities. Nuclear subcompartmentalization thus may provide a higher-order level for the regulation of biochemical activities of large T in vivo.

Adenosine Triphosphatases↗
PubMed1989

Only a minor fraction of plasma membrane-associated large T antigen in simian virus 40-transformed mouse tumor cells (mKSA) is exposed on the cell surface.

The bulk of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed cells localizes within the cell nucleus, while a minor fraction specifically associates with the plasma membrane (PM) and is exposed on the cell surface. PM-associated large T seems to span the lipid bilayer but, on the other hand, does not display typical features of a transmembrane protein. To further characterize the postulated transmembrane orientation of large T, we asked whether all large T molecules associated with the plasma membrane indeed are exposed on the cell surface. We compared the amount of cell surface-exposed large T, determined on living cells by a sensitive 3H-protein A-binding assay and by external immunoprecipitation, with that of total PM-associated large T extracted from isolated PM. We demonstrate that in mKSA cells (SV40-transformed BALB/c mouse fibroblasts), total PM-associated large T accounted for a substantial portion (ca. 2%) of total cellular large T. However, only 0.1 to 0.2% of it could be detected on the cell surface. Thus, only a minor fraction of PM-associated large T (less than 10%) is exposed on the surface of these cells. Interior PM-associated large T is stably associated with the plasma membrane, while the small fraction of surface-exposed large T is rapidly released from the cell surface.

Adenoviridae↗
PubMed1989

Cooperation of SV40 large T antigen and the cellular protein p53 in maintenance of cell transformation.

We analysed large T antigen expression and metabolic stabilisation of the cellular protein p53 in cells of a matched pair of SV40 tsA mutant (tsA58) N-type or A-type transformants, respectively. At the permissive growth temperature (32 degrees C), cells of both transformants, like SV40 wild-type transformed cells, were phenotypically transformed and expressed large T antigen, as well as metabolically stable p53 (both complexed and free p53). At the nonpermissive growth temperature (39 degrees C), cells of the N-type transformant reverted to a normal phenotype, whereas cells of the A-type transformant still displayed a transformed phenotype. Under these growth conditions, the mutant large T antigens in both cell types were no longer able to complex p53 (both in vivo and in vitro), but the metabolic stabilities of the free p53 in these cells correlated with their phenotypes: p53 in cells of the N-type transformant was rapidly degraded, whereas it was metabolically stable in cells of the A-type transformant. This difference in p53 stability correlated with an in vivo functional difference between the mutant large T antigens at the nonpermissive growth temperature: large T antigen in cells of the N-type transformant no longer stably associated with the cellular chromatin and the nuclear matrix, but accumulated in the nucleoplasm. In contrast, large T antigen in cells of the A-type transformant at least partially had retained this ability. Maintenance of SV40 cell transformation thus seems to require both a functional large T antigen and a metabolically stabilised p53.

Animals↗
PubMed1989

A novel mechanism for covalent attachment of fatty acid to SV40 large T antigen.

The plasma membrane associated subclass of simian virus 40 large T antigen is specifically acylated with palmitic acid in vivo. To further analyze possible biological functions of fatty acid acylation, we developed a target-bound cell free in vitro acylation assay, in which immunopurified large T, bound to protein A-sepharose, was incubated with [3H]fatty acid. In this assay, large T was efficiently labeled with [3H]palmitic acid, but not with [3H]myristic acid. Thus the specificity of the in vivo labeling was preserved in vitro, too. The specific acylation of large T in vitro seemed to occur by an autocatalytic reaction, since it was found to be independent of added acyltransferases and exogenous energy. The energy for this reaction must be provided by the large T molecule itself, probably by an energy-rich internal ester bond. Our results provide evidence for a novel mechanism for the covalent attachment of fatty acids to proteins, which might also operate in vivo.

Acylation↗
PubMed1988

A subclass of the adenovirus 72K DNA binding protein specifically associating with the cytoskeletal framework of the plasma membrane.

We have analyzed by immunofluorescence microscopy and by biochemical cell fractionation the subcellular distribution of the adenovirus type 2 72K DNA binding protein (DBP) during the course of infection in HeLa cells. Early in infection, the 72K DBP was strictly localized in the cell nucleus. However, as infection progressed, the 72K DBP was additionally found in other subcellular fractions, notably in association with the cytoskeletal framework of the plasma membrane, the plasma membrane lamina. Pulse-chase experiments demonstrated that this association was specific. Control experiments excluded the possibility of an artificial redistribution of the 72K DBP during cell fractionation. Our data, therefore, demonstrate that a significant portion of the 72K DBP during late times of infection associates specifically with the cytoskeletal framework of plasma membranes of infected cells.

Adenoviridae Infections↗
PubMed1988

Analysis of mechanisms controlling the interactions of SV40 large T antigen with the SV40 ORI region.

We have characterized the interactions of simian virus 40 (SV40) large tumor antigen (large T) with the control region of the SV40 genome, the SV40 ORI, by analyzing the specific binding of large T antigen to SV40 wild-type origin DNA and to isolated binding sites I and II, respectively. DNA binding affinities of large T antigen were determined under standardized conditions and DNA excess, using a target-bound DNA binding assay (M. Hinzpeter, E. Fanning, and W. Deppert, 1986, Virology 148, 159-167). Our results show that large T antigen exhibits similar affinities for isolated binding sites I and II and for combined sites I and II on wild-type ORI DNA. When the fraction of large T antigen molecules (calculated per large T antigen monomers) able to bind specifically to these sites was determined (DNA binding activity of large T antigen) we found that only 2% of large T antigen molecules present in extracts of lytically infected cells were able to bind to isolated site II, whereas about 50% bound to isolated site I. However, only about 10% of large T antigen molecules bound to the complete wild-type ORI, containing combined binding sites I and II. Thus, a much larger proportion of large T antigen molecules is capable of binding specifically to site I as is suggested by analysis of large T antigen binding to combined sites I and II on the SV40 wild-type ORI. These findings indicate that the interaction of large T antigen with the SV40 wild-type ORI is restricted on one hand by the ability of large T antigen to bind to site II, and on the other hand by the spatial arrangement of binding sites I and II on the SV40 wild-type ORI.

Adenosine Triphosphate↗
PubMed1988

DNA binding properties of murine p53.

We analysed the in vitro binding of p53 from normal (3T3) and from chemically transformed (Meth A) Balb/c mouse cells to double-stranded (ds-) DNA and to single-stranded (ss-) DNA by DNA-cellulose chromatography. We confirm previous findings that p53 in cellular extracts exhibits ds-DNA-binding activity (Lane and Gannon, 1983). In addition, we demonstrate that such p53 also binds to ss-DNA. Analyses with immunopurified p53 protein provide evidence that this DNA-binding activity is intrinsic to p53. DNA binding of p53 could not be inhibited by a monoclonal antibody specific for the C-terminal region. An N-terminal deletion mutant of p53 (Rovinski et al., 1987) exhibited similar DNA-binding properties as wild-type p53, indicating that the N-terminus also is dispensable for DNA binding. We further show a close correlation between the DNA-binding activity of p53 from 3T3 cells and its association with nuclear substructures.

Animals↗
PubMed1987

Specific interaction of simian virus 40 large T antigen with cellular chromatin and nuclear matrix during the course of infection.

We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.

Animals↗
PubMed1987

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.

Animals↗
PubMed1987

Analysis of biological and biochemical parameters for chromatin and nuclear matrix association of SV40 large T antigen in transformed cells.

We analysed biological and biochemical parameters for the association of the simian virus 40 (SV40) large tumor antigen (large T) with the cellular chromatin and the nuclear matrix in SV40-transformed cells. Nuclear subclasses of large T were isolated by in situ cell fractionation (Staufenbiel & Deppert, 1983) and first analysed for possible biological functions in the maintenance of cellular transformation. Like large T in SV40 wild-type transformants, large T in SV40 tsA mutant (tsA58)-transformed cells, expressing a temperature-dependent phenotype, was present in all nuclear subfractions (nucleoplasm, chromatin and nuclear matrix), when cells were kept at the growth temperature permissive for the expression of the transformed phenotype (32 degrees C). When tsA mutant-transformed cells were shifted to the non-permissive growth temperature (39 degrees C), they reverted to the normal phenotype. Concomitantly, large T lost its ability to associate with the cellular chromatin and the nuclear matrix, indicating that an association of large T with these subcellular structures may be important for the maintenance of cellular transformation. We next analysed the DNA-binding properties (sequence-specific binding to the SV40 origin of replication, ORI) of the nuclear subclasses of SV40 wild-type and of SV40 mutant large T defective in SV40 ORI binding in order to determine the influence of sequence-specific DNA binding on the association of large T with the chromatin and the nuclear matrix. Our detailed analyses show distinct differences in the ability of the various nuclear subclasses of large T to bind to the SV40 ORI, but suggest that the association of large T with the chromatin and the nuclear matrix is mediated by protein-protein interactions rather than by sequence-specific DNA binding.

Antigens, Viral, Tumor↗
PubMed1986

A new sensitive target-bound DNA binding assay for SV40 large T antigen.

We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI.

Antigens, Polyomavirus Transforming↗
PubMed1986

The kinase activity of SV40 large T antigen is mediated by a cellular kinase.

Large T antigen (large T) extracted from SV40-infected or transformed cells exhibits an in vitro protein kinase activity, whose origin and biological significance up to now had been obscure. We have addressed the questions of whether this activity is intrinsic to large T or arises by association with a cellular kinase, and, furthermore, whether this activity might play a biological role in vivo. Instead of analyzing large T from whole-cell lysates, where non-specific association of a cellular kinase(s) with large T might easily occur, we analyzed individual cellular subclasses of large T, isolated from their in vivo locations. In contrast to large T isolated from whole-cell lysates which was always kinase positive, none of the cellular subclasses of large T prepared by in situ fractionation of SV40-transformed mKSA cells exhibited detectable in vitro kinase activity. We could demonstrate that our fractionation conditions neither inactivated the large T-associated kinase activity nor dissociated it from large T when they were applied to kinase-positive large T isolated from whole-cell lysates. We conclude that large T does not contain an intrinsic kinase activity. This conclusion was further supported by our finding that it was possible to remove the large T-associated kinase activity from kinase-positive large T preparations and to reconstitute it by incubating the kinase-negative large T with cell lysates from various cell lines. Therefore, the simplest way of interpreting our results is that the in vitro kinase activity measured with large T preparations from whole-cell lysates is the result of an in vitro association of a cellular kinase(s) with large T during certain conditions of cell lysis.

Animals↗