scholarly journals Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

2011 ◽  
Vol 437 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Giuseppe Filomeni ◽  
Simone Cardaci ◽  
Ana Maria Da Costa Ferreira ◽  
Giuseppe Rotilio ◽  
Maria Rosa Ciriolo
Keyword(s):  
Protein Kinase ◽  
Cortical Neurons ◽  
Reverse Genetics ◽  
Combined Treatment ◽  
Pentose Phosphate ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Induced Apoptosis ◽  
Amp Activated Protein Kinase

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N′]copper(II), named [Cu(isaepy)2], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38MAPK (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38MAPK and p53. Similarly, reverse genetics of p38MAPK yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38MAPK/p53. Fuel supplies counteracted [Cu(isaepy)2]-induced apoptosis and AMPK/p38MAPK/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)2] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38MAPK/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)2] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)2] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

scholarly journals UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53

2002 ◽  
Vol 365 (1) ◽  
pp. 133-145 ◽  
Author(s):  
Nadine CHOUINARD ◽  
Kristoffer VALERIE ◽  
Mahmoud ROUABHIA ◽  
Jacques HUOT
Keyword(s):  
Adaptive Response ◽  
Human Keratinocytes ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Normal Human Keratinocytes ◽  
Mitogen Activated Protein ◽  
Normal Human ◽  
Induced Apoptosis ◽  
Negative Mutant

Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

AMPK, MAPK and Bax in the heart: some questions answered

2008 ◽  
Vol 412 (2) ◽  
pp. e15-e16 ◽  
Author(s):  
Vilmante Borutaite
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Wide Field ◽  
Biochemical Journal ◽  
Mitogen Activated Protein ◽  
Isolated Cardiac Myocytes ◽  
And Migration ◽  
Induced Apoptosis ◽  
Amp Activated Protein Kinase ◽  
Rapid Activation

The question of how Bax is activated during apoptosis to perform its role in permeabilization of mitochondrial membranes is intriguing for investigators in the wide field of cell death research. In their paper published in the Biochemical Journal in 2006, Capano and Crompton presented their discovery that simulated ischaemia causes rapid activation of AMPK (AMP-activated protein kinase) which phosphorylates and activates p38 MAPK (mitogen-activated protein kinase) leading to Bax activation and translocation to mitochondria in isolated cardiac myocytes. This was the first report on the molecular mechanism of Bax activation and migration during ischaemia-induced apoptosis in cardiomyocytes.

Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
Author(s):  
Brian K. Dieckgraefe ◽  
Danielle M. Weems
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Erk Activation ◽  
Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

scholarly journals Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells

2005 ◽  
Vol 393 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Natalia Makeeva ◽  
Jason W. Myers ◽  
Nils Welsh
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Nitric Oxide Donor ◽  
Cell Leukaemia ◽  
Dominant Negative Mutant

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced β-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2′-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38α-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCδ (protein kinase Cδ), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1β and interferon-γ, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.

scholarly journals Coordinate Regulation of IκB Kinases by Mitogen-Activated Protein Kinase Kinase Kinase 1 and NF-κB-Inducing Kinase

1998 ◽  
Vol 18 (12) ◽  
pp. 7336-7343 ◽  
Author(s):  
Shino Nemoto ◽  
Joseph A. DiDonato ◽  
Anning Lin
Keyword(s):  
Protein Kinase ◽  
Interleukin 1 ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ikk Complex ◽  
Mitogen Activated Protein ◽  
Protein Kinase Kinase

ABSTRACT IκB kinases (IKKα and IKKβ) are key components of the IKK complex that mediates activation of the transcription factor NF-κB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-κB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-α) and interleukin-1 and can potentiate the stimulatory effect of TNF-α on IKK and NF-κB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-α. MEKK1 interacts with and stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

Role of the atypical protein kinase Cζ in regulation of 5′-AMP-activated protein kinase in cardiac and skeletal muscle

2009 ◽  
Vol 297 (2) ◽  
pp. E349-E357 ◽  
Author(s):  
John R. Ussher ◽  
Jagdip S. Jaswal ◽  
Cory S. Wagg ◽  
Heather E. Armstrong ◽  
David G. Lopaschuk ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Minor Role ◽  
Atypical Protein Kinase ◽  
Downstream Target ◽  
Ampk Phosphorylation ◽  
Amp Activated Protein Kinase

During metabolic stress, phosphorylation and activation of 5′-AMP-activated protein kinase (AMPK) becomes a major regulator of cellular energy metabolism in heart and skeletal muscle. Despite this, the upstream regulation of AMPK in both heart and muscle is poorly understood. Recent work has implicated the atypical protein kinase Cζ (PKCζ) as a regulator of AMPK in endothelial cells via phosphorylation of LKB1, an upstream AMPK kinase (AMPKK). Our goal was to determine the potential role PKCζ plays in regulating AMPK in cardiac and skeletal muscle. Cultures of H9c2 myocytes (cardiac) and C2C12 myotubes (skeletal muscle) were pretreated with a selective PKCζ pseudosubstrate peptide inhibitor and treated with various AMPK activating agents to determine whether PKCζ regulates AMPK. PKCζ activity was also examined in isolated working rat hearts subjected to ischemia. We show that PKCζ is not involved in regulating threonine 172 AMPK phosphorylation induced by metformin or phenformin in either cardiac or skeletal muscle cells but is involved in 5-aminoimidazole-4-carboxamine-1-β-d-ribofuranoside (AICAR)-induced AMPK phosphorylation in cardiac muscle cells. Activation of PKCζ with high palmitate concentrations is also insufficient to increase AMPK phosphorylation. Furthermore, we show that the ischemia-induced activation of AMPK is not accompanied by increased PKCζ activity. Finally, we show that PKCζ may actually be a downstream target of AMPK in skeletal muscle, since adenoviral expression of a dominant-negative mutant of AMPK prevented metformin- and AICAR-induced phosphorylation of PKCζ. We conclude that PKCζ plays a very minor role in the regulation of AMPK in cardiac and skeletal muscle and may actually be a downstream target of AMPK in skeletal muscle.

Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway

2006 ◽  
Vol 290 (2) ◽  
pp. L291-L297 ◽  
Author(s):  
Min Ding ◽  
Chuanshu Huang ◽  
Yongju Lu ◽  
Linda Bowman ◽  
Vince Castranova ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Crystalline Silica ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
P38 Kinase ◽  
Kinase C ◽  
Pkc Inhibitors

Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previous studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKCα and PKCε from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKCα or PKCε markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKCα and PKCε are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway.

scholarly journals Immature and Mature Cortical Neurons Engage Different Apoptotic Mechanisms Involving Caspase-3 and the Mitogen-Activated Protein Kinase Pathway

2002 ◽  
Vol 22 (8) ◽  
pp. 935-950 ◽  
Author(s):  
Christian Lesuisse ◽  
Lee J. Martin
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Cortical Neurons ◽  
Caspase 3 ◽  
Mitogen Activated Protein Kinase ◽  
Immature Neurons ◽  
Mitogen Activated Protein ◽  
Mature Neurons ◽  
Induced Apoptosis ◽  
Map Kinase Kinase

The authors used cultured mouse cortical neurons to study mechanisms of DNA damage-induced apoptosis in immature and mature neurons. Neurons were maintained viably for 60 days in vitro (DIV60). The increased levels of glutamate receptors, synaptic proteins, and glycolytic enzyme were used to track maturation. Exposure of neurons to the DNA-damaging agent camptothecin induced apoptosis in immature (DIV5) and mature (DIV25–30) neurons. Internucleosomal fragmentation of DNA emerged more rapidly in mature neurons than in immature neurons. Immunoblotting revealed that cleaved caspase-3 increased in apoptotic DIV5 neurons but not in DIV30 neurons, but immunolocalization showed accumulation of cleaved caspase-3 in DIV5 and DIV30 neurons. A reversible caspase-3 inhibitor blocked apoptosis in DIV5 neurons but not in DIV30 neurons. Phosphorylation of extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAP kinase)-42/44 occurred preapoptotically in mature but not immature neurons, while Erk54 nuclear translocation and MAP kinase kinase kinase-1 cleavage into putative caspase-3–generated proapoptotic fragments occurred in DIV5 but not DIV30 neurons. Inhibition of Erk activation with MAP kinase kinase inhibitor blocked apoptosis at both ages. The results show that immature and mature cortical neurons engage different signaling mechanisms in MAP kinase and caspase pathways during apoptosis; thus, neuron age influences the mechanisms and progression of apoptosis.

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