Limits...
DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function.

Schlegel CR, Georgiou ML, Misterek MB, Stöcker S, Chater ER, Munro CE, Pardo OE, Seckl MJ, Costa-Pereira AP - Cell Death Dis (2015)

Bottom Line: Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family.Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor.RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, ICTEM, Du Cane Road, London W12 0NN, UK.

ABSTRACT
Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.

Show MeSH

Related in: MedlinePlus

The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. (a) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS (b–g) and A549 (h–m) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS (b) and A549 cells (h). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS (c) and A549 cells (i). Forty-eight hours after siRNA transfection, NAD+, NADH, NADP+ and NADPH levels were analysed using colorimetric assays in U2OS (d–g) and A549 cells (j–m). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t-test (paired, one tailed) (*P<0.05, **P<0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4385915&req=5

fig5: The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. (a) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS (b–g) and A549 (h–m) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS (b) and A549 cells (h). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS (c) and A549 cells (i). Forty-eight hours after siRNA transfection, NAD+, NADH, NADP+ and NADPH levels were analysed using colorimetric assays in U2OS (d–g) and A549 cells (j–m). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t-test (paired, one tailed) (*P<0.05, **P<0.01)

Mentions: Eukaryotic cells use two key metabolic pathways for ATP generation (Figure 5a). Both pathways start with glycolysis as the first step of glucose metabolism, converting one glucose molecule into two molecules of pyruvate with gain of two ATP molecules. Aerobic respiration involves the transport of pyruvate into the mitochondria and mitochondrial respiration downstream of glycolysis through the tricarboxylic acid (TCA)/Krebs cycle, which yields another thirty four ATP molecules/molecule of glucose.26 Anaerobic respiration includes glycolysis and the fermentation of pyruvate to lactate, a metabolic pathway that bypasses mitochondrial respiration, and is predominantly upregulated in cancer cells (Warburg effect), which cancer cells use to produce most of their energy.18, 27


DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function.

Schlegel CR, Georgiou ML, Misterek MB, Stöcker S, Chater ER, Munro CE, Pardo OE, Seckl MJ, Costa-Pereira AP - Cell Death Dis (2015)

The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. (a) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS (b–g) and A549 (h–m) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS (b) and A549 cells (h). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS (c) and A549 cells (i). Forty-eight hours after siRNA transfection, NAD+, NADH, NADP+ and NADPH levels were analysed using colorimetric assays in U2OS (d–g) and A549 cells (j–m). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t-test (paired, one tailed) (*P<0.05, **P<0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385915&req=5

fig5: The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. (a) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS (b–g) and A549 (h–m) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS (b) and A549 cells (h). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS (c) and A549 cells (i). Forty-eight hours after siRNA transfection, NAD+, NADH, NADP+ and NADPH levels were analysed using colorimetric assays in U2OS (d–g) and A549 cells (j–m). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t-test (paired, one tailed) (*P<0.05, **P<0.01)
Mentions: Eukaryotic cells use two key metabolic pathways for ATP generation (Figure 5a). Both pathways start with glycolysis as the first step of glucose metabolism, converting one glucose molecule into two molecules of pyruvate with gain of two ATP molecules. Aerobic respiration involves the transport of pyruvate into the mitochondria and mitochondrial respiration downstream of glycolysis through the tricarboxylic acid (TCA)/Krebs cycle, which yields another thirty four ATP molecules/molecule of glucose.26 Anaerobic respiration includes glycolysis and the fermentation of pyruvate to lactate, a metabolic pathway that bypasses mitochondrial respiration, and is predominantly upregulated in cancer cells (Warburg effect), which cancer cells use to produce most of their energy.18, 27

Bottom Line: Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family.Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor.RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, ICTEM, Du Cane Road, London W12 0NN, UK.

ABSTRACT
Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.

Show MeSH
Related in: MedlinePlus