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E T Coffey

Publications and source records attributed to E T Coffey.

12 recordsLinked to original sources
PubMed2001

Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-beta isoform.

In this study we report the activation of c-Jun N-terminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin-mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c-Jun. The activation of JNK occurs through activation of mitogen-activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK-directed phosphatases. We have previously shown that overexpression of the HSF2-beta isoform inhibits the activation of HSF2 upon hemin-induced erythroid differentiation. Here we demonstrate that HSF2-beta overexpression blocks the hemin-induced activation of the MKK4-JNK pathway, suggesting an erythroid lineage-specific JNK activation likely to be regulated by HSF2.

Anisomycin↗
PubMed2000

Dual roles for c-Jun N-terminal kinase in developmental and stress responses in cerebellar granule neurons.

c-Jun N-terminal kinases (JNKs) typically respond strongly to stress, are implicated in brain development, and are believed to mediate neuronal apoptosis. Surprisingly, however, JNK does not respond characteristically to stress in cultured cerebellar granule (CBG) neurons, a widely exploited CNS model for studies of death and development, despite the regulation of its substrate c-Jun. To understand this anomaly, we characterized JNK regulation in CBG neurons. We find that the specific activity of CBG JNK is elevated considerably above that from neuron-like cell lines (SH-SY5Y, PC12); however, similar elevated activities are found in brain extracts. This activity does not result from cellular stress because the stress-activated protein kinase p38 is not activated. We identify a minor stress-sensitive pool of JNK that translocates with mitogen-activated protein kinase kinase-4 (MKK4) into the nucleus. However, the major pool of total activity is cytoplasmic, residing largely in the neurites, suggesting a non-nuclear role for JNK in neurons. A third JNK pool is colocalized with MKK7 in the nucleus, and specific activities of both increase during neuritogenesis, nuclear JNK activity increasing 10-fold, whereas c-Jun expression and activity decrease. A role for JNK during differentiation is supported by modulation of neuritic architecture after expression of dominant inhibitory regulators of the JNK pathway. Channeling of JNK signaling away from c-Jun during differentiation is consistent with the presence in the nucleus of the JNK/MKK7 scaffold protein JNK-interacting protein, which inhibits JNK-c-Jun interaction. We propose a model in which distinct pools of JNK serve different functions, providing a basis for understanding multifunctional JNK signaling in differentiating neurons.

Animals↗
PubMed1999

The mechanism of Ara-C-induced apoptosis of differentiating cerebellar granule neurons.

Neurotoxicity is one of the side-effects of the therapeutically useful antitumour agent, Ara-C (or 1-beta-d-arabinofuranosyl-cytosine, cytarabine). This agent is also reported to induce cell death of cultured neurons. In this study, we show that Ara-C-induced death of differentiating rat cerebellar granule neurons is prevented by cycloheximide at concentrations corresponding to its action in preventing protein synthesis. The death is accompanied by cleavage of the caspase substrate poly ADP ribose polymerase (PARP) and c-Abl-dependent activation of the stress-activated protein kinases c-Jun N-terminal kinase and p38. However, c-Jun levels do not rise and the activation of the stress-activated protein kinases is not required for this form of neuronal death. Cyclin-dependent kinase (cdk) activity and inappropriate cell-cycle re-entry have been implicated in some forms of death in differentiated neurons. Here we show that Ara-C-induced death of cerebellar granule neurons is prevented by an inhibitor of cdk4, whereas inhibition of cdk1, -2 and -5 mimics the death, and non-cdk4/6 cdks are inhibited by Ara-C treatment. Cdk1 and -2 are dramatically down-regulated during neuronal differentiation, and neither Ara-C nor inhibition of these cdks induces death in mature neurons. This mechanism could also play a significant role in the neurotoxicity associated with the therapeutic use of Ara-C, as cdk levels can be upregulated in stressed neurons of adult brain. We propose that the balance between cdk4/6 and cdk1/2/5 activity may determine the survival of early differentiating neurons, and that DNA-damaging agents may induce neuronal death by inhibiting cdk1/2/5 under conditions which require these activities for survival.

Androstadienes↗
PubMed1998

Suppression of Fas/APO-1-mediated apoptosis by mitogen-activated kinase signaling.

Jurkat T cells undergo rapid apoptosis upon stimulation of the Fas/APO-1 (CD95) receptor. We examined the role of the mitogen-activated protein kinase (MAPK) cascade as a negative regulator of Fas-mediated apoptosis. To this end, we used both physiologic and artificial activators of MAPK, all of which activate MAPK by distinct routes. MAPK activity could be efficiently elevated by two T cell mitogens, the lectin PHA and an agonistic Ab to the T cell receptor complex as well as by the type 1 and 2A phosphatase inhibitor, calyculin A, and the protein kinase C-activating phorbol ester, tetradecanoyl phorbol acetate. All these treatments were effective in preventing the characteristic early and late features of Fas-mediated apoptosis, including activation of caspases. Our results indicate that the elevated MAPK activities intervene upstream of caspase activation. The degree of MAPK activation by the different stimuli used in our study corresponds well to their potency to inhibit apoptosis, indicating that MAPK activation serves as an efficient modulator of Fas-mediated apoptosis. The role of MAPK in modulation of Fas-mediated apoptosis was further corroborated by transient transfection with constitutively active MAPK kinase, resulting in complete inhibition of the Fas response, whereas transfection with a dominant negative form of MAPK kinase had no effect. Furthermore, the apoptosis inhibitory effect of the MAPK activators could be abolished by the specific MAPK kinase inhibitor PD 098059. Modulation of Fas responses by MAPK signaling may determine the persistence of an immune response and may explain the insensitivity of recently activated T cells to Fas receptor stimulation.

Apoptosis↗
PubMed1997

Detection of myelin basic protein isoforms by organic concentration.

An effective technique was developed, which allowed rapid isolation of highly pure myelin basic protein (MBP) including its distinct isoforms. The procedure employs homogenization of central nervous system (CNS) tissue in chloroform, which specifically extracts MBP. Subsequently, methanol was used to convert the protein susceptible to quantitative transfer into the acidic aqueous phase. MBP was purified from bovine, chicken, fish, human, guinea-pig, mouse, rabbit, rat, and swine brains. Analysis on SDS-PAGE and immunoblotting using polyclonal MBP-specific serum recognized proteins corresponding to the sizes of previously identified MBP isoforms of 21.5, 18.5, 17.2, and 14.2 kDa and three predicted isoforms of 20.2, 16.0, and 13 kDa. The MBP obtained was readily soluble in water and possessed the capacity to induce experimental autoimmune encephalomyelitis in susceptible mice. The protein was also suitable for use as a substrate for protein kinases.

Animals↗
PubMed1997

Neurotrophins protect cultured cerebellar granule neurons against the early phase of cell death by a two-component mechanism.

Cerebellar granule neurons cultured with serum develop a mature neuronal phenotype, including stimulus-coupled release of glutamate, and depend on elevated potassium for survival. We find that cells cultured with serum undergo two phases of cell death. By 6 d in vitro, 30-50% of the cells present are dead; after this time the remaining cells die. Elevated potassium prevents only this later phase of death, whereas neurotrophins protect these cells against the early phase of death. Factors that bind p75(NTR) or TNF-R, members of the same receptor family, exhibit voltage-sensitive calcium channel-dependent protection, whereas ligands of expressed Trk receptors show additional calcium channel-independent protection. The cells express TrkB protein and show elevated c-Fos and c-Jun levels in response to BDNF. No TrkA is detected, although p75(NTR) protein is expressed and NGF induces depolarization-dependent elevation of c-Jun levels. In the presence of the protein kinase C inhibitor bisindolylmaleimide, BDNF-induced survival promotion is reduced partially, whereas NGF-induced death is unmasked. Basal survival mechanisms are insensitive to inhibition of PK-C or PI-3 kinase. We conclude that BDNF promotes survival in part via its TrkB receptor, whereas there is an additional pathway promoting survival and elevating c-Jun evoked by both NGF and BDNF via a non-Trk receptor.

Androstadienes↗
PubMed1997

Brain derived neurotrophic factor induces a rapid upregulation of synaptophysin and tau proteins via the neurotrophin receptor TrkB in rat cerebellar granule cells.

We have examined the effects of neurotrophins brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on the expression of the maturation-specific proteins synaptophysin and tau, and the growth-associated protein (GAP)-43 in cerebellar granule cells. We find that BDNF but not NGF rapidly (within 2 h) upregulates levels of synaptophysin, tau and c-Fos correlating with expression of the neurotrophin receptor TrkB. The rapid increase in synaptophysin is not preceded by c-Fos elevation suggesting a post-transcriptional mechanism may be involved. In contrast, no upregulation of GAP-43 levels are seen within this time period. Phorbol ester mimics the effects of BDNF, indicating that protein kinase C (PKC) is either a component of, or feeds into the signalling mechanism. We conclude that BDNF, characterized to be survival promoting early in differentiation of cerebellar granule cells, enhances maturation at a later stage.

Animals↗
PubMed1994

Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga-GI sensitive Ca2+ channel which is coupled to glutamate exocytosis.

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga-GI completely blocks KCl-evoked glutamate exocytosis but only partially inhibits KCl-evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl-induced Ca2+ entry. Aga-GI completely attenuates KCl-induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga-GI.

Animals↗
PubMed1994

Glutamate exocytosis and MARCKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically activated by diacylglycerol and arachidonic acid.

4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K+ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4 beta-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] mimics the action of 4 beta-phorbol dibutyrate, but only in the presence of 2 microM arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1S,3R)-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca2+ channel or Ca(2+)-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1S,3R)-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1S,3R)-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.

Animals↗
PubMed1993

Protein kinase C and the regulation of glutamate exocytosis from cerebrocortical synaptosomes.

The role of protein kinase C (PKC) in the regulation of transmitter glutamate release from rat cerebral cortical synaptosomes is investigated. Two depolarization protocols are used: first, elevated KCl, which produces a clamped depolarization, and second, 4-aminopyridine, which evokes spontaneous "action potentials" allowing any potential modulation of Na+ or K+ channels to influence release. Although the PKC inhibitor Ro 31-8220 prevents both the depolarization-evoked and phorbol dibutyrate (PDBu)-evoked phosphorylation of the major presynaptic PKC substrate, myristoylated alanine-rich C kinase substrate, it is without effect on KCl-evoked Ca(2+)-dependent glutamate release. Ro 31-8220 totally inhibits the Ca(2+)-dependent 4-aminopyridine-evoked release of glutamate in the presence and absence of PDBu and again decreases the phosphorylation of myristoylated alanine-rich C kinase substrate. Ro 31-8220 strongly inhibits the 4-aminopyridine-evoked increase in [Ca2+] both in the presence and absence of PDBu and antagonizes the PDBu enhancement of depolarization. This indicates that PKC isoforms activatable by PDBu and sensitive to Ro 31-8220 play no discernable role in Ca(2+)-secretion coupling per se in cerebral cortical glutamatergic nerve terminals, but that the kinase plays a major role in regulating the depolarization of the terminal.

4-Aminopyridine↗