Biomedical subjects

D J Burns

Publications and source records attributed to D J Burns.

At least 37 records · Page 2Linked to original sources

PubMed1994

Presence of a beta II protein kinase C-selective nuclear membrane activation factor in human leukemia cells.

In human promyelocytic (HL60) leukemia cells beta II protein kinase C (PKC) is selectively translocated to the nucleus in response to proliferative stimuli. At the nucleus, beta II PKC directly phosphorylates the nuclear envelope polypeptide lamin B at two consensus PKC phosphorylation sites, Ser395 and Ser405. Phosphorylation of these sites by beta II PKC leads to solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro (Hocevar, B.A., Burns, D.J., and Fields, A.P. (1993) J. Biol. Chem. 268, 7545-7552). We have now investigated the molecular basis for beta II PKC-selective nuclear translocation and lamin B phosphorylation using an in vitro reconstitution system. We find that beta II PKC phosphorylates nuclear envelope lamin B at 10-20 times the rate of alpha PKC, whereas both kinases phosphorylate soluble lamin B at similar rates. Comparative tryptic phosphopeptide analysis demonstrates that alpha PKC and beta II PKC phosphorylate identical sites, Ser395 and Ser405, on soluble lamin B. These data suggest that a component(s) of the nuclear envelope confers beta II PKC-selective nuclear activation and lamin B phosphorylation. Extraction of nuclear envelopes with either non-ionic detergent (2% n-octyl glucoside) or organic solvent (CHCl3/CH3OH/H2O; 10:10:3) abolishes beta II PKC-selective phosphorylation of nuclear lamin B. Nuclear membrane extracts reconstitute beta II PKC-selective phosphorylation, indicating the presence of a beta II PKC-selective nuclear membrane activation factor (NMAF). NMAF selectively activates beta II PKC histone H1 kinase activity 3-4-fold above the level achieved with optimal concentrations of Ca2+, diacylglycerol, and phosphatidylserine. Finally, NMAF activity is not affected by exhaustive protease treatment, suggesting that it is a nuclear membrane lipid(s) or lipid metabolite. These data suggest that NMAF plays a physiologic role in the nuclear activation of beta II PKC.

Base Sequence↗

PubMed1994

Identification of nuclear beta II protein kinase C as a mitotic lamin kinase.

Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we investigate the involvement of two proposed human mitotic lamin kinases, beta II protein kinase C (PKC) and p34cdc2/cyclin B kinase, in human lamin B1 phosphorylation in vitro and in intact cells. We find that both kinases can phosphorylate purified soluble lamin B at similar rates. However, beta II PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34cdc2/cyclin B kinase. beta II PKC-mediated phosphorylation of lamin B is confined to two sites, Ser395 and Ser405, within the carboxyl-terminal domain, whereas human p34cdc2/cyclin B kinase phosphorylates a single site, Ser23, in the amino-terminal domain. A second potential p34cdc2/cyclin B kinase site within the carboxyl-terminal domain, Ser393, is not phosphorylated by human p34cdc2/cyclin B kinase. However, invertebrate p34cdc2/cyclin B kinase from sea star exhibits a different specificity, phosphorylating both amino- and carboxyl-terminal sites. Mitotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide mapping demonstrates that the beta II PKC site, Ser405, is a prominent target of mitotic lamin B phosphorylation in vivo. beta II PKC translocates to the nucleus during the G2/M phase of cell cycle concomitant with phosphorylation of Ser405, indicating a physiologic role for nuclear beta II PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-terminal domain of mitotic lamin B which are not phosphorylated by either beta II PKC or p34cdc2/cyclin B kinase suggests the involvement of other lamin kinase(s) in G2/M phase lamin B phosphorylation.

Animals↗

PubMed1994

Phospholipase D activation in fibroblast membranes by the alpha and beta isoforms of protein kinase C.

The regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) by protein kinase C (PRC) in membranes of Chinese hamster lung fibroblasts (CCL39) was studied using conventional PKC isoforms alpha, beta and gamma isolated from rat brain and recombinant PKC isoforms. Cells were incubated with [14C]choline to label endogenous phosphatidylcholine before membranes were prepared and assayed for release of [14C]choline. PKC alpha was the most potent activator of PLD, producing a maximal effect at approximately 0.1 microgram/ml. PKC beta also stimulated PLD but was less potent and less efficacious, whereas PKC gamma was ineffective. Stimulation required addition of a PKC activator, but the isoform specificity was the same whether phorbol 12-myristate 13-acetate (PMA) or Ca2+ was used. Recombinant Ca(2+)-independent PKC isoforms delta, epsilon, and zeta failed to stimulate PLD, but recombinant PKC beta 1 stimulated PLD in a manner similar to the purified brain PKC beta. Immunoblot analysis of the soluble fraction of CCL 39 fibroblasts detected only the alpha and zeta isoforms of PKC. The results suggest that PKC alpha and beta are activators of PLD and that PKC alpha is responsible for the activation in these fibroblasts.

Animals↗

PubMed1994

The production of an active protein kinase C-delta in insect cells is greatly enhanced by the use of the basic protein promoter.

Occasionally, only a small percentage of recombinant proteins produced in the baculovirus expression system are functionally active. We had previously shown that the majority of protein kinase C-delta (PKC-delta) produced in insect cells was inactive; less than 1% of the expressed enzyme had lipid-dependent kinase activity. In this report, we have attempted to optimize the production of a catalytically active PKC-delta. Under optimum conditions, we were able to increase the levels of PKC-delta from 10-20% to about 65% of the total cellular protein; however, there was no increase in the levels of catalytically active enzyme. Expression of PKC-delta as a fusion protein or as a secreted protein also met with limited success. Under all conditions, expression of PKC-delta proteins under control of the strong polyhedrin promoter resulted in the production of large amounts of inactive enzyme. Expression under the control of the basic protein promoter, Pcor, resulted in the reduction of the levels of recombinant protein by a factor of about four, but the PKC-delta enzyme produced under these conditions was 10- to 15-fold more active. Thus, the earlier temporal expression of PKC-delta in insect cells resulted in the production of more active enzyme.

Animals↗

PubMed1994

Cyclic adenosine 3',5'-monophosphate-binding proteins in human ovarian cancers.

The aims of the present study were to characterise an assay for cAMP-binding proteins in ovarian cancer and then to measure levels in a series of tumours with a view to developing a potential prognostic indicator for this disease. Levels and types of binding proteins have been measured in cytosols from 50 ovarian tumours. Binding proteins were detected in all tumours but, as calculated from Scatchard analysis, binding levels ranged from 267 to 12,037 fmol per mg of cytosol protein (mean value of 4248 fmol mg-1). Dissociation constants of binding varied between 0.4 x 10(-8) and 5.9 x 10(-8) (mean value 2.3 x 10(-8)). Types of binding protein were detected by incubation with the photoaffinity ligand 8-N3-[32P]cAMP, followed by polyacrylamide gel electrophoresis and autoradiography. Labelled proteins with molecular weights of 52, 48, 43, 39 and 37 kDa were identified in the cytosols. The proportion and pattern of bands detected varied between different cytosols. The significance of these findings awaits clinical follow-up of the patients.

Adenine Nucleotides↗

PubMed1994

Translocation and downregulation of protein kinase C isoenzymes-alpha and -epsilon by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts.

Protein kinase C isoenzymes can be subdivided into two classes, based on their requirement for calcium. Protein kinase C-alpha, beta I, -beta II, and -gamma are calcium dependent, whereas protein kinase C-gamma, -epsilon, -zeta, -eta, and -theta are calcium independent. We have examined the expression, translocation, downregulation, and activation of calcium-dependent and -independent protein kinase C isoenzymes in human skin keratinocytes and fibroblasts. Human keratinocytes and fibroblasts expressed protein kinase C-alpha, -delta, -epsilon, and -zeta mRNA and protein, whereas protein kinase C-eta (L) was detected only in keratinocytes. Protein kinase C-beta I, -beta II, -gamma, and -theta were not detected in either cell type. The protein kinase C activators 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1 (50 nM, for 5 min) induced translocation of protein kinase C-alpha and -epsilon cytosol to membrane in both keratinocytes and fibroblasts. 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1, for 18 h, induced complete downregulation (i.e., loss) of protein kinase C-alpha and -epsilon in keratinocytes, but only partial downregulation was observed in fibroblasts. The subcellular distribution of protein kinase C-delta, -zeta or protein kinase C-eta, in keratinocytes or fibroblasts, did not change in response to 12-0-tetradecanoylphorbol 13-acetate or bryostatin-1. These data indicate differential expression, subcellular distribution, and regulation of protein kinase C isoenzymes in human skin cells.

Base Sequence↗

PubMed1993

The cDNA sequence encoding human protein kinase C-zeta.

A 1779-bp complementary DNA (cDNA) that encodes protein kinase C-zeta (PKC-zeta) has been isolated from a human frontal cortex library using traditional plaque-screening methods and PCR screening. The deduced 592-amino-acid sequence of the human PKC-zeta clone has a 95-96% identity to those deduced from the previously described rat and mouse PKC-zeta clones.

Amino Acid Sequence↗

PubMed1993

Molecular and biochemical characterization of a recombinant human PKC-delta family member.

Two cDNA clones coding for the human protein kinase C-delta (PKC-delta) were fortuitously isolated during the process of screening a human library for a cDNA clone of an unrelated protein, the nucleolar protein fibrillarin. The two human homologues have about 88% nucleotide sequence identity to the rat and mouse PKC-delta cDNA clones. A comparison of the predicted amino acid sequences of the two human PKC-delta clones with the rat and mouse homologues indicated a greater degree of sequence divergence (89-90% homology) compared to the high degree of sequence conservation observed with other human PKC family members and their mammalian counterparts. Expression of the clones in the baculovirus insect-cell expression system indicated that both proteins exhibited phorbol ester binding activity, and were dependent upon phosphatidylserine and diacylglycerol for maximal activation. Further characterization of the properties of the human PKC-delta revealed substrate and lipid dependencies distinct from other members of the protein kinase C family; including PKC-deltas isolated from other species. The dissimilarities in the predicted amino acid sequences between the human and other mammalian species could account in part for some of these observed biochemical differences.

Amino Acid Sequence↗

PubMed1993

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation.

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.

Base Sequence↗

PubMed1993

Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics.

Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the beta II PKC isotype is selectively translocated and activated at the nucleus. We recently identified the nuclear envelope component lamin B1 as a major substrate for nuclear PKC both in whole cells and in vitro. Using highly purified human beta II PKC and isolated nuclear envelopes from the human promyelocytic (HL60) leukemia cell line, we have now determined the major sites for beta II PKC-mediated lamin B phosphorylation. Using a combination of cyanogen bromide cleavage, direct microsequencing, tryptic phosphopeptide, and phosphate release analyses, two major sites of PKC-mediated phosphorylation, Ser395 and Ser405, have been identified. These sites lie within the carboxyl-terminal domain of lamin B immediately adjacent to the central alpha-helical rod domain. Functionally, beta II PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro. beta II PKC-mediated lamin B phosphorylation is inhibited by 1) a monoclonal antibody directed against the active site of PKC, 2) a PKC pseudosubstrate inhibitor peptide, and 3) a PKC peptide substrate. Two observations indicate that PKC-mediated lamin B phosphorylation and solubilization is due to direct phosphorylation of lamin B by PKC rather than indirect activation of a cdc2 kinase. Neither immunodepletion with p13suc1 Sepharose beads nor the presence of a p34cdc2 kinase peptide substrate had any effect on PKC-mediated lamin B phosphorylation. Therefore, we conclude that beta II PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear beta II PKC, like p34cdc2 kinase, may function to regulate nuclear lamina structural stability during cell cycle.

Amino Acid Sequence↗

PubMed1993

Fruit developmental regulation of the kiwifruit actinidin promoter is conserved in transgenic petunia plants.

We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5'-flanking region (nucleotides -1301 to +58) of a kiwifruit actinidin gene was fused to the beta-glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides -115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.

Base Sequence↗

PubMed1993

The mere exposure effect for stimuli presented below recognition threshold: a failure to replicate.

Repeated exposure to a stimulus increases one's affect toward that stimulus. This finding, termed the mere exposure effect, has been obtained even when the exposure durations are below the threshold of recognition. Reported are three experiments designed to assess whether the mere exposure effect for stimuli presented below the threshold of recognition could be obtained for meaningful stimuli. Analysis showed that meaningfulness of the stimuli had no effect on ratings of affect; however, we did not replicate previous findings of a mere exposure effect for stimuli that were not recognized. The robustness of the mere exposure effect for stimuli presented below the threshold of awareness was questioned.

Adult↗

PubMed1993

The effects of generation on item and order retention in immediate and delayed recall.

Recent research has shown that generating words from fragments, relative to simply reading them, inhibits processing of order information. Nairne, Riegler, and Serra (1991) showed that this reduction in processing of order information leads to deficits in recall performance. In three experiments, we generally replicate Nairne et al.'s results and demonstrate that the deficit in recall for the generated items is dependent on the easy distractor task and the relatively short (30-sec) retention interval they used. When a difficult distractor task was used, generating produced a deficit in amount of order information processed, but actually facilitated recall when recall was delayed 80 sec. The results are consistent with the hypothesis that generating words inhibits order processing, but they do not support the contention that the reduction in order processing is responsible for the deficit in recall that is sometimes observed for the generated items. The importance of the item-order distinction in explaining the generation effect, as well as the role of the item-order distinction in the long-term-memory arena, is questioned.

Adolescent↗

PubMed1993

Examining a processing tradeoff explanation of proactive interference.

Burns (1989) claims that proactive interference effects occur in paired-associate learning because of tradeoffs in relational and response-specific processing. Consistent with this claim, Burns demonstrated that free recall of critical-list responses is better in the interference condition than in the control condition. Burns's processing tradeoff explanation predicts that the occurrence of this reverse-interference effect should be positively correlated with the occurrence of traditional interference effects. We present several experiments whose results are inconsistent with this prediction. We hypothesize that the reverse-interference effect is a list-length effect. The results of a final experiment, contrasting the predictions of the list-length and processing tradeoff explanations, support the list-length explanation.

Adult↗

PubMed1993

A comparison of large-scale Sf9 insect cell growth and protein production: stirred vessel vs. airlift.

A simple and relatively inexpensive stirred-vessel system for the large-scale growth of insect cells (Sf9) is described. Sf9 cell growth in a stirred-vessel fermentor and an airlift fermentor were compared on the basis of maximum cell density and average population doubling time. Also, both fermentor systems were compared with respect to the large-scale production of a recombinant human protein (protein kinase C-eta). No significant differences in Sf9 cell growth or protein expression levels were apparent between the two fermentor systems. However, large differences in cost and scale-up of each system are discussed with respect to the large-scale production of recombinant proteins.

Animals↗

PubMed1992

Sequence and expression of human protein kinase C-epsilon.

Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.

Amino Acid Sequence↗

PubMed1992

Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase in diacylglycerol.

The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Protein kinase C contains two phorbol ester binding domains.

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.

Amino Acid Sequence↗