Biomedical subjects

P E Branton

Publications and source records attributed to P E Branton.

At least 55 records · Page 3Linked to original sources

PubMed1995

Induction of p53-independent apoptosis by hygromycin B: suppression by Bcl-2 and adenovirus E1B 19-kDa protein.

Hygromycin B, an aminoglycoside antibiotic that is widely used to establish stable mammalian cell lines that carry a bacterial gene conferring resistance to the drug, is shown here to induce apoptotic programmed cell death in susceptible cells. Dying cells exhibited typical features of apoptosis, including cell shrinkage, membrane blebbing, nuclear pyknosis, and extensive internucleosomal fragmentation of DNA. Employing concentrations of hygromycin B that are typically used for selecting stable cell lines, we show that susceptible cells die rapidly, exhibiting the morphological properties of apoptosis by 18 h and detectable DNA fragmentation as early as 2 h after receiving the drug. G418, on the other hand, required days to cause cell death, which was not accompanied by internucleosomal DNA fragmentation. Apoptotic cell killing by hygromycin B did not require expression of wild-type p53 and was suppressed by both Bcl-2 and the Adenovirus type 5 E1B 19-kDa protein.

Adenovirus E1B Proteins↗

PubMed1994

Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus.

Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death. It contains a COOH-terminal signal anchor sequence that is selective for import and insertion of Bcl-2 into the mitochondrial outer membrane and, by a different mechanism, can also direct the protein to other membrane sites. Deletion of the signal anchor sequence rendered Bcl-2 cytosolic and impaired its ability to prevent apoptotic death of human KB cells infected with a mutant form of adenovirus type 5 that does not make E1B 19-kDa protein. When the predicted transmembrane domain of the Bcl-2 signal anchor was replaced with that of the signal anchor of the yeast outer mitochondrial membrane protein, Mas70p, the Bcl-2/Mas70p hybrid was found to be very similar to Bcl-2 in its distribution within transfected KB cells, in its ability to heterodimerize with Bax, and in its ability to suppress apoptosis. These results are consistent with a model in which the transmembrane segment contributes to the function of Bcl-2 by targeting and anchoring the protein to strategic membrane locations in the cell. Concentration of Bcl-2 at these sites may contribute to its proposed role as regulator, or component, of an antioxidant pathway.

Adenovirus E1B Proteins↗

PubMed1994

Characterization of the 55K adenovirus type 5 E1B product and related proteins.

In addition to major proteins of 19K and 55K (176 and 496 residues, 176R and 496R, respectively), early region 1B (E1B) of human adenovirus type 5 (Ad5) is predicted to encode at least three other polypeptides of 156R, 93R and 84R that share 79 amino-terminal residues with 496R. We have used a series of specific antipeptide sera to identify and partially characterize these proteins. 84R was produced in large amounts, 156R somewhat less, and 93R at very low levels. Synthesis of 176R, 496R, as well as the E2A 72K DNA-binding protein commenced shortly after that of E1A proteins in Ad5-infected KB cells. Production of 156R, 93R and 84R began somewhat later, but prior to the synthesis of the late structural protein IX and hexon. 156R, which is composed of the 79 amino-terminal and 77 carboxy-terminal amino acids of 496R, migrated on SDS-PAGE as two species which appeared to differ by their degree of phosphorylation. 156R and 496R yielded identical tryptic phosphopeptides that contained both phosphoserine and phosphothreonine, and one of these was immunoprecipitated by a serum specific for the carboxy terminus. These results suggested that Ser-490 and/or Ser-491 as well as Thr-495 are major sites of phosphorylation in these proteins.

Adenovirus E1B Proteins↗

PubMed1994

Functional importance of complex formation between the retinoblastoma tumor suppressor family and adenovirus E1A proteins as determined by mutational analysis of E1A conserved region 2.

Adenovirus early region 1A (E1A) products induce DNA synthesis, transform primary rodent cells, and activate transcription factor E2F through complex formation with an array of cellular proteins via the E1A amino terminus and conserved regions 1 and 2 (CR1 and CR2). Interactions with the retinoblastoma tumor suppressor, pRb, and related proteins p107 and p130 rely somewhat on CR1 but largely on CR2, which contains a core binding sequence Leu-122-X-Cys-X-Glu. We introduced point mutations in CR2 to define such interactions more precisely. In human cells, alteration of any of the conserved residues within the binding core eliminated complex formation with pRb. Conversion of nonconserved Thr-123 to Pro (but not to either Ala or Ser) disrupted binding of pRb, presumably because of conformational changes in the binding core. No single E1A point mutant was completely defective in binding p107, suggesting that molecular interactions between E1A proteins and p107 clearly differ from those with pRb and p130. In general, the patterns of complex formation by E1A mutants in rat, monkey, and human cells were quite similar. All mutants which failed to bind significant amounts of pRb also failed to transform primary rat cells. Several mutants demonstrated selective binding to pRb, p107, and p130, but transforming activity corresponded largely with complex formation with pRb, regardless of the levels of interactions with p107 and p130. Mutants defective for binding of both pRb and p107 failed to induce the activity of transcription factor E2F; however, quite high levels were activated by E1A mutants that interacted with p107 alone. These results suggested that both pRb and p107 are important regulators of E2F activity but that complex formation with and activation of E2F by p107 are insufficient for cell transformation.

Adenovirus E1A Proteins↗

PubMed1994

Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation.

Adenovirus E1B Proteins↗

PubMed1994

Functional interactions within adenovirus E1A protein complexes.

The transforming potential of adenovirus E1A oncogene products derives largely from the formation of complexes with cellular proteins, including the p105Rb tumor suppressor and a related p107 species, p130 and p300 proteins, and cyclin A (p60cycA). Extensive quantitative analyses using E1A deletion mutants identified unique binding patterns for each of these polypeptides within the amino terminus and conserved regions 1 and 2 (CR1 and CR2) of E1A proteins. A novel protein, termed p400, was found by peptide mapping to be related to p300, and, like p300, to require the E1A amino terminus and a portion of CR1 for binding. p130 was shown to be related to p107, and like p107, to associate with p60cycA. p107, p130 and p105Rb all interacted primarily with CR2, however, sequences within CR1 and the amino terminus were capable of weak interactions and appeared to function cooperatively with CR2 to bind these proteins. Protein kinase activity present in E1A complexes probably derives at least in part from p60cycA-linked p33cdk2 associated with p107 and p130. In vitro phosphorylation of complexes purified by immunoprecipitation resulted in labeling of several proteins. p60cycA was phosphorylated to about the same extent in cyclin A complexes prepared from either AD5- or mock-infected KB cells, however, that of p130 and p107 was dramatically higher in p60cycA complexes from infected cells. p300 was also phosphorylated in complexes prepared using E1A-specific antibodies. Thus one role of E1A proteins in signal transduction and regulation of the cell cycle may be to control the biological activity of p107, p130 and p300 by enhancing their phosphorylation through complex formation.

Adenovirus E1A Proteins↗

PubMed1993

Role of phosphorylation near the amino terminus of adenovirus type 5 early region 1A proteins.

Human adenovirus early region 1A (E1A) proteins act as transcriptional regulators and function in the control of DNA synthesis and cell transformation. Little is known about how these viral products are functionally regulated. E1A proteins of adenovirus serotype 5 (Ad5) are phosphorylated at several serine residues and previous studies had indicated that both Ser-89 and Ser-219 are substrates for one or more of the cdc2 family of cell cycle kinases. A second residue near the amino terminus, Ser-96, may also be a site. Although phosphorylation of Ser-89 causes a major shift in gel mobility, the effect on E1A biological activity is unclear. In the present studies we have shown by mutational analysis that phosphorylation at Ser-89 also regulates phosphorylation at Ser-96, suggesting that the gel mobility shift is the result of multiple phosphorylation events. Phosphorylation at Ser-89 did not seem to affect E1A-mediated repression of the simian virus 40 enhancer or trans-activation of the E3 promoter significantly, but it did appear to have a modest but significant effect on transformation of primary baby rat kidney cells.

Adenovirus E1A Proteins↗

PubMed1993

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

3T3 Cells↗

PubMed1993

Isolation of chicken phosphotyrosyl phosphatase cDNA sequences and identification of a brain-specific species related to human PTP zeta.

The first example of a chicken cDNA sequence encoding a phosphotyrosyl phosphatase (PTPase) has been identified and found to contain coding sequences for the entire cytoplasmic and membrane spanning domains as well as a portion of the extracellular region of a transmembrane PTPase resembling human PTP zeta. Like HPTP zeta, chicken PTP zeta contained two phosphatase domains (D1 and D2), and D2 lacked a critical cysteine residue required for catalytic activity. The entire intracellular portion of CPTP zeta was expressed in bacteria and shown to be capable of dephosphorylating both p-nitrophenylphosphate and reduced carboxyamidomethylated and maleyated lysozyme but not phosphoseryl casein. Genetic analysis indicated that the presence of D2 was required for full activity. CPTP zeta mRNA was identified as a single large transcript expressed exclusively in the brain of chick embryos at both early and late stages of embryogenesis. These results suggested that CPTP zeta may perform a brain-specific function and have a role in development.

Amino Acid Sequence↗

PubMed1992

Adenovirus type 12 early region 1B proteins and metabolism of early viral mRNAs.

Early region 1B (E1B) of human adenoviruses encodes two major proteins. The 19-kDa polypeptide appears to prevent E1A-induced cytolysis and DNA degradation. The larger E1B product of approximately 55 kDa, which is essential for viral replication, plays a role in the accumulation and stability of viral mRNAs and the late shutoff of host metabolism. For serotype 12 (Ad12), this 482-residue (482R) protein is essential for viral DNA replication. In the present report we have used a series of mutants to examine the roles of Ad12 482R and the 19-kDa, 163R protein in the metabolism of early viral mRNAs. No specific effects on the accumulation of early (or late) mRNAs were detected with any of the mutants affecting 163R. With mutant dl42, which encodes an altered 482R product that lacks residues 114-155, both viral DNA replication and late viral protein synthesis were defective. Accumulation of E1A transcripts in the nucleus and cytoplasm resembled wt. The levels of mRNAs from early regions E1B, E2A and E3 at later times during infection were somewhat lower than those of wt, but this decrease may have been due to the absence of progeny viral DNA in dl42-infected cells. However, the accumulation of both E2B and E4 mRNAs at all times was severely reduced. These data suggested that the requirement of 482R for Ad12 DNA replication may be related to its specific role in the metabolism of E2B and E4 mRNAs that encode products necessary for viral DNA synthesis.

Adenovirus E1B Proteins↗

PubMed1992

Phosphorylation of adenovirus E1A proteins by the p34cdc2 protein kinase.

Adenovirus early region 1A (E1A) products are phosphorylated nuclear oncoproteins which appear to derive transforming activity largely through interactions with cellular proteins including the tumor suppressor p105/Rb-1 and cyclin A (p60cycA), a regulatory subunit associated with p34cdc2 and the related protein kinase p33cdk2. We have identified several sites of phosphorylation on E1A proteins previously and showed that phosphorylation at Ser-89 alters electrophoretic mobility significantly and affects E1A-mediated transforming activity to some extent. We now report that both Ser-89 and Ser-219, the major E1A phosphorylation site, were phosphorylated in vitro by p34cdc2 purified from HeLa cells. We also found that E1A proteins seemed to be phosphorylated at the highest levels in vivo in mitotic cells which express maximal levels of p34cdc2 kinase activity. Thus, in addition to forming complexes with p60cycA, a regulator of p34cdc2 and related kinases, and p105/Rb-1 which exhibits cell cycle-dependent phosphorylation, E1A proteins seem to be substrates for p34cdc2. These data suggested that a link could exist between phosphorylation, cell cycle progression, and the regulation of transforming activity of E1A proteins.

Adenovirus Early Proteins↗

PubMed1992

Overexpression of the E1B 55-kilodalton (482R) protein of human adenovirus type 12 appears to permit efficient transformation of primary baby rat kidney cells in the absence of the E1B 19-kilodalton protein.

To analyze the structure and function of the E1B 19,000-molecular-weight protein (19K protein) (163R) of human adenovirus type 12, mutants were produced at various positions across the 163R-coding sequence. Viruses bearing mutations within the first 100 or so amino acids yielded unstable 163R-related products, induced DNA degradation and enhanced cytopathic effect (cyt/deg phenotype) in KB cells, and transformed primary rodent cells at much lower efficiencies than wild-type (wt) virus. Deletion of the final 16 residues at the carboxy terminus had no phenotypic effect. Alteration of residue 105 reduced transforming efficiency significantly, suggesting that this region of 163R is functionally important. Disruption of the AUG initiation codon at nucleotide 1542 blocked production of 163R completely but resulted in higher levels of E1B 55K-482R protein synthesis and a transforming efficiency similar to that of wt virus. These data suggested that while 163R is of some importance, normal transforming efficiencies can be obtained in its absence if 482R is overexpressed.

Adenovirus Early Proteins↗

PubMed1991

Individual adenovirus E1B proteins induce transformation independently but by additive pathways.

The specific contributions of human adenovirus type 5 early region 1B (E1B) proteins were examined using mutants which synthesize these products individually. In cooperation with E1A, transformation of primary baby rat kidney cells was achieved with either the 176R protein or 496R protein alone, albeit at an efficiency considerably less than that observed when both were present. These results indicate that transformation mediated by either E1B product can proceed independently, but that the processes involved are additive.

Adenovirus Early Proteins↗

PubMed1990

Retinoblastoma growth suppressor and a 300-kDa protein appear to regulate cellular DNA synthesis.

Previous work has suggested that oncogenic transformation by the E1A gene products of adenovirus type 5 may be mediated through interactions with at least two cellular proteins, the 105-kDa product of the retinoblastoma growth suppressor gene (p105-Rb) and a 300-kDa protein (p300). By using viral mutants, we now show that the induction of cellular DNA synthesis in quiescent cells by E1A differs from transformation in that E1A products induce synthesis if they are able to bind to either p105-Rb or p300, and only mutant products that bind to neither are extremely defective. These results suggest that p105-Rb and p300 (or cellular proteins with similar E1A-binding properties) provide parallel means by which DNA synthesis can be regulated.

Adenovirus Early Proteins↗

PubMed1989

Analysis of phosphorylation sites in the exon 1 region of E1A proteins of human adenovirus type 5.

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces five mRNAs that encode proteins of 55, 171, 217, 243, and 289 residues. We have shown previously that the major products of 289 and 243 residues are phosphorylated at a minimum of three sites of which one, Ser-89, is located in the amino terminal half of the protein. In the present report we show that these E1A proteins are also phosphorylated at a second site in this region located at Ser-96. The 171 and 217 residue E1A species were also tentatively identified and, as predicted, neither contained the Ser-89 or Ser-96 sites but both appeared to be phosphorylated at the same sites as 289R and 243R toward the carboxy terminus. Studies with mutants in which Ser-89 or Ser-96 were converted to alanine residues indicated that phosphorylation of Ser-89 but not Ser-96 induces the major shift in gel mobility of E1A products. However, neither site appears to be of major importance in the regulation of E1A-mediated transactivation or transformation.

Acid Phosphatase↗

PubMed1989

Mapping of a phosphorylation site in the 176R (19 kDa) early region 1B protein of human adenovirus type 5.

The 176-residue (176R) early region 1B (E1B) protein of human adenovirus type 5 (Ad5) was shown to be phosphorylated at serine in lytically infected KB cells at a level estimated to be about one phosphate group per 28 176R molecules. Through the analysis of peptides generated by cleavage with cyanogen bromide and Staphylococcus aureus V-8 protease the phosphorylation site was mapped to Ser-164. Using site-directed mutagenesis, a mutant was produced in which the codon for Ser-164 was changed to that of asparagine while leaving the coding sequence for the overlapping 496R protein unchanged. This virus, which replicated well on human KB cells, produced normal levels of 176R, but in an unphosphorylated form. The mutant transformed baby rat kidney cells in cooperation with E1A at an efficiency about one-half that obtained with wt E1B. These data therefore gave little indication that phosphorylation is essential for the function of 176R.

Adenovirus Early Proteins↗

PubMed1989

Phosphorylation at serine 89 induces a shift in gel mobility but has little effect on the function of adenovirus type 5 E1A proteins.

Phosphorylation at serine 89 was shown to be the major cause of the shift in gel migration of the 289R and 243R early region 1A (E1A) proteins of human adenovirus type 5. However, conversion of Ser-89 to alanine by site-directed mutagenesis did not abolish E1A transactivating or transforming activities, suggesting that phosphorylation at this site is not necessary for these E1A functions.

Adenovirus Early Proteins↗

PubMed1989

Binding of the Rb1 protein to E1A products is required for adenovirus transformation.

Several cellular proteins, including polypeptides of 300, 107 and 105kDa, associate with the products of the E1A gene of adenovirus type 5. Here we show that the 105kDa species is the product of the recessive oncogene Rb1 which is absent or altered in retinoblastomas and other human cancers. About 75% of the total Rb1 protein in infected human KB cells was found to be complexed with E1A products and these data support the hypothesis that E1A-mediated transformation results from the elimination of functional Rb1 protein within the cell. However, while the interaction of E1A products with this cellular protein is necessary, transformation appears also to require binding of the 300 and 107kDa polypeptides. We also found that both the Rb1 protein and the 107kDa E1A-binding species were undetectable in a retinoblastoma cell line lacking both alleles of the Rb1 gene. These data suggest that the absence of both the 107kDa and 105kDa/Rb1 proteins may be involved in the development of the oncogenic phenotype of retinoblastoma cells.

Adenovirus Early Proteins↗