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Biomedical subjects

W Eckhart

Publications and source records attributed to W Eckhart.

At least 19 recordsLinked to original sources
PubMed1992

Induction of c-jun protooncogene expression and transcription factor AP-1 activity by the polyoma virus middle-sized tumor antigen.

Polyoma virus middle-sized tumor (PymT) antigen is required for neoplastic cell transformation by polyoma virus. We studied changes in gene expression accompanying expression of PymT in murine fibroblasts. These experiments showed that PymT differentially affects several growth-related genes. c-jun protooncogene expression was highly increased, whereas the expression of two growth arrest-specific genes (gas) was reduced, in cells transformed by PymT. Cotransfection experiments showed that the increase in c-jun expression resulted from elevated activity of the transcription factor AP-1 and was mediated through the phorbol 12-tetradecanoate 13-acetate response element in the c-jun promoter. The degree of c-Jun/AP-1 activation by different PymT mutants correlated with their transforming capability, suggesting that regulation of c-Jun/AP-1 activity may play a role in cell transformation by polyoma virus.

Animals↗
PubMed1992

Phosphorylation sites in the amino-terminal region of mouse p53.

Phosphorylation is an attractive mechanism for regulating the functions of p53. The p34cdc2 kinase, which is involved in regulation of the cell cycle, phosphorylates serine-315 of human p53 in vitro. Casein kinase II phosphorylates serine-389 of mouse p53 in vitro. The amino-terminal region of mouse p53 contains a cluster of potential serine phosphorylation sites. Those sites have been proposed to be sites for phosphorylation by a double-stranded DNA-dependent kinase (DNA-PK) from HeLa cells and can be dephosphorylated by protein phosphatase 2A. To identify in vivo phosphorylation sites in the amino-terminal region of mouse p53, we mutated potential phosphorylation sites and analyzed the mutant proteins by tryptic phosphopeptide mapping. We identified serine-7, -9, -18, and -37 as in vivo phosphorylation sites. We further showed that mouse p53 expressed in bacteria is phosphorylated by DNA-PK on amino-terminal serine residues in vitro.

Amino Acid Sequence↗
PubMed1992

Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

Animals↗
PubMed1990

The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II.

The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.

Amino Acid Sequence↗
PubMed1990

Activation of the pp60c-src protein kinase is an early event in colonic carcinogenesis.

Colonic neoplasia provides an opportunity to study tumor progression because most carcinomas arise from adenomas (polyps), which, in turn, arise from normal epithelia. The malignant potential of adenomas varies with size, histology, and degree of dysplasia. Polyps that are less than 2 cm with villous architecture and severe dysplasia are most likely to contain carcinoma. Previous studies demonstrated that the in vitro protein-tyrosine kinase activity of pp60c-src from colon carcinomas is significantly higher than that from adjacent normal mucosa. Here we report that the protein kinase activity of pp60c-src is also elevated in colonic polyps. Activity is highest in malignant polyps and in greater than 2-cm benign polyps that contain villous structure and severe dysplasia. Thus, pp60c-src activation occurs in benign polyps that are at greatest risk for developing cancer. These data suggest that activation of the protooncogene product pp60c-src may be an important event in the genesis of human colon carcinoma.

Antibodies, Monoclonal↗
PubMed1990

Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo.

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.

Animals↗
PubMed1990

Transcriptional regulation of early-response genes during polyomavirus infection.

Infection of quiescent BALB/c 3T3 cells with polyomavirus leads to the biphasic accumulation of RNA from several early-response genes. The steady-state levels of RNA of c-fos, c-myc, and c-jun were detected at 0 to 1 and 12 to 30 h after infection but not at 6 h postinfection. Infections with mutant viruses suggest that in the context of a virus infection with other tumor (T) antigens present, large T and middle T antigens are dispensable for the effect and small t antigen is important for the regulation, perhaps in conjunction with large T or middle T antigens. These results are in agreement with those of Zullo et al. (Proc. Natl. Acad. Sci. USA 84:1210-1214). We have further found that this regulation occurs in NIH 3T3 cells and primary mouse embryo fibroblasts. DNA synthesis is not required for this effect. Half-lives of c-fos, c-myc, and c-jun were similarly short at both early and late times after infection, as determined by dactinomycin chase. The regulation of expression occurs at the transcriptional level. Nuclear run-on experiments showed increased rates of transcription both early and late after infection. Also, the polyomavirus early region can transactivate the c-fos promoter in transient transfection assays.

Animals↗
PubMed1989

Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53.

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.

Adenosine Triphosphatases↗
PubMed1989

pp60c-src activation in human colon carcinoma.

We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).

Carcinoma↗
PubMed1988

pp60c-src expression in the developing rat brain.

We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.

Aging↗
PubMed1988

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.

Animals↗
PubMed1988

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.

Amino Acid Sequence↗
PubMed1987

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain.

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.

Animals↗
PubMed1987

cDNA cloning with a retrovirus expression vector: generation of a pp60c-src cDNA clone.

We used a murine retroviral expression vector, containing a genomic clone of the chicken c-src gene, a bacterial origin of replication, and a selectable marker, to remove 10 introns from the c-src gene. All 10 introns were removed accurately, and no mutations were introduced. The processed gene encoded a functional pp60c-src protein tyrosine kinase.

Cloning, Molecular↗
PubMed1987

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.

Animals↗
PubMed1986

Expression of the polyoma middle-size T antigen in Escherichia coli.

We constructed a plasmid encoding a hybrid protein, consisting of the N-terminal signal sequence of the major outer membrane lipoprotein (lpp) of Serratia marcescens joined to the polyoma middle-size T antigen (mT antigen). The hybrid protein expressed under the control of a lpp-lac hybrid promoter was synthesized at levels up to 5% of newly synthesized protein and could be accumulated in Escherichia coli strains carrying the Cap R mutation. The mT antigen produced in E. coli was precipitated by polyoma antitumor serum, and by serum directed against a synthetic peptide corresponding to the C terminus of the authentic mT antigen. The protein was secreted into the periplasmic space, from which it could be released by osmotic shock. The bacterial mT antigen had no detectable associated protein kinase activity.

Antigens, Viral, Tumor↗