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Biomedical subjects

P E Branton

Publications and source records attributed to P E Branton.

At least 37 records · Page 2Linked to original sources
PubMed1998

The early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death by apoptosis.

Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.

Adenovirus E4 Proteins↗
PubMed1997

p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

Amino Acid Sequence↗
PubMed1997

Characterization of an E2F-p130 complex formed during growth arrest.

Electrophoretic mobility shift assays were used to analyse the pattern of E2F transcription factor complexes containing pRB and related 'pocket' proteins associated with changes in growth of monkey CV-1 cells. Little change was noted in pRB/E2F complexes following growth arrest or serum stimulation. Serum starvation induced the formation of a novel slowly-migrating p130/E2F complex, termed C7, which was comparable to one reported previously in terminally differentiated C2C12 mouse cells (Corbeil et al., 1995, Oncogene 11, 909-920) and thought to contain one or more additional unidentified proteins. After serum stimulation, C7 complex disappeared in S-phase but returned during mitosis. A major E2F complex containing p107 appeared during S-phase but was undetectable at other times. It appeared likely that regulation of pRB, p107 and p130 occurs by several mechanisms. First, phosphorylation has previously been proposed to be involved. All pRB-related proteins were found to be highly phosphorylated during S-phase when E2F activity was highest, and hypophosphorylated in arrested cells when E2F activity was low. Second, the absolute levels of some members of the RB family varied during the cell cycle. Levels of pRB protein and pRB/E2F complexes changed little; however, the amounts of both p107/E2F and p130/E2F complexes varied according to the overall levels of p107 and p130 proteins. Formation of the novel C7 complex in serum arrested cells suggested a third regulatory mechanism mediated by interactions with additional proteins. Our studies indicated that binding of such proteins with p130/E2F occurred via a site in the 'pocket' similar to that utilized by adenovirus E1A proteins. Such proteins could function as additional regulators of E2F-driven transcription.

Adenovirus E1A Proteins↗
PubMed1997

Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis.

It has been known for some time that expression of the 243-residue (243R) human adenovirus type 5 (Ad5) early region 1A (E1A) protein causes an increase in the level of the cellular tumor suppressor p53 and induction of p53-dependent apoptosis. Deletion of a portion of conserved region 1 (CR1) had been shown to prevent apoptosis, suggesting that binding of p300 and/or the pRB retinoblastoma tumor suppressor and related proteins might be implicated. To examine the mechanism of the E1A-induced accumulation of p53, cells were infected with viruses expressing E1A-243R containing various deletions which have well-characterized effects on p300 and pRB binding. It was found that in human HeLa cells and rodent cells, complex formation with p300 but not pRB was required for the rise in p53 levels. However, in other human cell lines, including MRC-5 cells, E1A proteins which were able to form complexes with either p300 or pRB induced a significant increase in p53 levels. Only E1A mutants defective in binding both classes of proteins were unable to stimulate p53 accumulation. This same pattern was also apparent in p53-null mouse cells coinfected by Ad5 mutants and an adenovirus vector expressing either wild-type or mutant human p53 under a cytomegalovirus promoter, indicating that the difference in importance of pRB binding may relate to differences between rodent and human p53 expression. The increase in p53 levels correlated well with the induction of apoptosis and, as shown previously, with the stimulation of cellular DNA synthesis. Thus, it is possible that the accumulation of p53 is induced by the induction of unscheduled DNA synthesis by E1A proteins and that increased levels of p53 then activate cell death pathways.

Adenovirus E1A Proteins↗
PubMed1997

Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.

Adenovirus E1A Proteins↗
PubMed1997

Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5.

The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E1A gene products to induce cell transformation. E1A proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is believed that the role of the E1B-55kDa protein in transformation is to protect against p53-dependent apoptosis by binding to and inactivating p53. It has been shown previously that the 55-kDa polypeptide abrogates p53-mediated transactivation and that mutants defective in p53 binding are unable to cooperate with E1A in transformation. We have previously mapped phosphorylation sites near the carboxy terminus of the E1B-55kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues greatly reduced transforming activity, and although the mutant 55-kDa protein was found to interact with p53 at normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now report that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to block E1A-induced p53-dependent apoptosis, whereas cells infected by mutant pm490/1/5A, which contains alanine residues at all three phosphorylation sites, exhibited extensive DNA fragmentation and classic apoptotic cell death. The E1B-55kDa product has been shown to exhibit intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absence of p53. Such repression activity was totally absent with mutant pm490/1/5A. These data suggested that inhibition of p53-dependent apoptosis may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carboxy terminus.

Adenovirus E1B Proteins↗
PubMed1997

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells.

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.

Adenovirus E1B Proteins↗
PubMed1996

Adenovirus E1B 19-kDa death suppressor protein interacts with Bax but not with Bad.

Adenovirus E1B 19-kDa protein (19K) is a member of the Bcl-2 family of suppressors of apoptosis. The suppressors function through heterodimerization with the death promoters, Bax and related proteins, thus establishing a set point within the cell that determines whether or not apoptosis is executed in response to a death signal. Sequence similarities between 19K and Bcl-2 are largely restricted to short Bcl-2 homology (BH) domains that mediate interaction with Bax. The BH1 sequence in 19K is degenerate but nevertheless contains a conserved glycine residue found in all family members that when mutated to alanine in Bcl-2 results in loss of Bcl-2 function and ability to dimerize with Bax (Yin, X.-M., Oltvai, Z. N., and Korsmeyer, S. J. (1994) Nature 369, 321-323). Here, we show that the analogous mutation in BH1 of 19K also abrogates the anti-apoptotic properties of 19K and its ability to interact with Bax, thus establishing the critical importance of this residue within BH1 and the likely similarity of Bcl-2 and 19K function. In distinct contrast to Bcl-2, however, 19K interaction was not detected with Bad, a Bcl-2/Bcl-XL dimerizing protein that can potentially regulate a Bax middle dotBcl-2/Bcl-XL survival set point and reinstate susceptibility to a death signal. Furthermore, the anti-apoptotic function of 19K was not overcome by enforced expression of Bad in transfected cells. This feature of 19K may provide adenovirus with a selective advantage in evading premature induction of apoptosis by the host cell.

Adenovirus E1B Proteins↗
PubMed1996

An activated epidermal growth factor receptor/Lck chimera restores early T cell receptor-mediated calcium response in a CD45-deficient T cell line.

In T cells, cell surface expression of CD45, a transmembrane tyrosine phosphatase, is required for T cell receptor (TCR) signal transduction. Indirect evidence suggests that CD45 function in TCR signaling involves the dephosphorylation of the C-terminal negative regulatory site of p56(lck), Tyr-505. To evaluate the importance of CD45-mediated dephosphorylation of p56(lck) Tyr-505 in TCR signaling, we established CD45(-) Jurkat cell lines expressing various forms of a chimera containing the extracellular and transmembrane domains of the epidermal growth factor receptor (EGFR) fused to p56(lck). We report that an activated EGFR/Lck chimera is able to reconstitute a Ca2+ response after CD3 stimulation in the absence of CD45 expression. In addition, the wild-type and kinase inactive versions of the EGFR/Lck chimera fail to restore early signaling. Restoration of the response by EGFR/LckF505 required EGF binding to the chimeric kinase. Altogether, these results provide the first direct evidence that the lack of efficient dephosphorylation of p56(lck) Tyr-505 is, in part, responsible for the unresponsiveness of CD45(-) cells. They also indicate that a second event is required for p56(lck) function in TCR signaling in addition to its dephosphorylation at Tyr-505.

Calcium↗
PubMed1996

Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins.

Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser209) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.

3T3 Cells↗
PubMed1996

Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase.

The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32.

Adenovirus E1A Proteins↗
PubMed1996

Association of CD45 with Lck and components of the Ras signalling pathway in pervanadate-treated mouse T-cell lines.

Pervanadate treatment of a mouse T-cell hybridoma cell line overexpressing an activated form of p56lck was shown to result in tyrosine phosphorylation of CD45. Immunoprecipitates prepared under mild lysis conditions using antibodies against CD45 contained a number of other proteins, including p56lck, that were not evident in the absence of pervanadate treatment or in T cells lacking activated Lck, implying that under these conditions, CD45 is present within complexes containing Lck and other proteins. Analyses involving deletion mutants of p56lck indicated that interactions with CD45 did not absolutely require the SH2 and SH3 regions of Lck. Three proteins of the Ras signalling pathway were also shown to associated with CD45: the GTPase-activating protein for Ras (rasGAP), the signalling protein Grb2, and, possibly via complex formation with Grb2, the guanine nucleotide exchange factor mammalian son of sevenless (mSOS). In addition, CD45 was also found in immunoprecipitates prepared from these cells using an antiserum which recognizes Vav. It is possible that rasGAP, Grb2 and Vav bind to phosphotyrosine residues on CD45 via SH2 domains, and such interactions may be specific as other SH2-containing proteins, including phospholipase C alpha (PLC gamma), the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). She and Syp/PTP1D were not detectably associated with CD45 under the same conditions. These data suggested that in addition to its role as a protein tyrosine phosphatase, CD45 may participate in T-cell activation by serving as a membrane docking site for components of the Ras signalling pathway.

Animals↗
PubMed1996

Importance of the Ser-132 phosphorylation site in cell transformation and apoptosis induced by the adenovirus type 5 E1A protein.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.

Adenoviridae↗
PubMed1996

Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis.

In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis.

3T3 Cells↗
PubMed1996

Expression of p53 in Saos-2 osteosarcoma cells induces apoptosis which can be inhibited by Bcl-2 or the adenovirus E1B-55 kDa protein.

Studies were carried out to determine the effects of introducing p53 using an adenovirus gene transfer vector into p53 null human Saos-2 osteogenic carcinoma cells. Expression of p53 led to cell death within 30-40 h. The morphology of these cells as determined by electron microscopy indicated that death was by apoptosis. Such death was significantly reduced in Saos-2 variants that express high levels of the Bcl-2 suppressor of apoptosis. It was also found that the E1B-55 kDa protein of human adenovirus type 5, which was known to bind and inactivate p53, blocks Saos-2 cell death following expression of p53. These results thus directly demonstrate that this viral protein is able to inhibit p53-induced apoptosis.

Adenovirus E1B Proteins↗
PubMed1995

Characterization of transcription factor E2F complexes during muscle and neuronal differentiation.

The activities of E2F transcription factors are inhibited by interactions with members of the retinoblastoma (RB) tumor suppressor family, p105RB, p107 and p130. In cycling cells p107 and p130 also interact with heterodimers comprised of Cdk2 and either A or E cyclins. We characterized E2F complexes present in C2C12 and P19 mouse cells induced to differentiate into muscle and neuronal cells, respectively. In both undifferentiated C2C12 and P19 cells, in addition to free species, E2F was found in complexes containing p107 or p130 and Cdk2. No E2F-pRB complexes were detected by electrophoretic mobility shift assays even though such cells were shown to contain pRB and E2F species capable of interacting in vitro. These results suggested that although present, pRB was unable to interact with E2F. Following differentiation of C2C12 cells into myotubes, E2F was present in at least two complexes which contained p130, but not in those containing p107 or Cdk2. Low levels of E2F-pRB complexes were also detected in fully differentiated C2C12 myotubes and in freshly isolated skeletal muscle. In the case of differentiated P19 neuronal cells, E2F was found in complexes containing pRB, p107 and p130. However, such cells may not be representative of fully differentiated neurons, as studies with rodent brain extracts indicated that only pRB-E2F complexes and those recognized by a p130-specific serum were present. These results suggested that in both muscle and neurons, pRB and p130 may play specific roles in the development or maintenance of terminal differentiation.

Animals↗
PubMed1995

Adenovirus E1A proteins induce apoptosis by both p53-dependent and p53-independent mechanisms.

E1A of human adenovirus type 5 (Ad5) encodes proteins of 289 and 243 residues (289R and 243R) which differ only by the 46 amino acid CR3 region known to activate expression of certain cellular and early viral genes. E1A proteins also induce DNA synthesis and cell transformation, but as well can stimulate apoptosis. Two adenovirus E1B products act to protect cells from E1A-induced cell death, including a 19 kDa protein which is functionally similar to the cellular Bcl-2 suppressor of apoptosis, and a 55 kDa species which binds to and inhibits p53. Previous studies suggested that E1A-induced cell death occurs via a p53-dependent mechanism requiring regions of E1A proteins linked to induction of DNA synthesis and cell transformation. We report here that the 289R E1A protein induces apoptosis in cell lines lacking p53, whereas the 243R product was dependent upon p53. We also show that this p53-independent process involves the expression of one or more additional viral proteins which are presumably synthesized in response to transactivation by 289R. Thus E1A proteins induce cell death by both p53-dependent and p53-independent mechanisms involving separate E1A functions.

Adenovirus E1A Proteins↗