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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.

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Genetic ablation of DAPK2 increases the rate of spontaneous mitochondrial membrane depolarisation using the JC-1 probe. U2OS (a–f) and A549 (g–m) cells were transfected with either siNS or siDAPK2. Forty-eight hours after transfection cells were incubated with JC-1 and the fluorescence of J-aggregates or monomers was measured in red and green fluorescence channels by flow cytometry. CCCP (50 μM) was used to induce complete mitochondrial depolarisation (c and i) and to set appropriate gates in U2OS (a–c) and A549 cells (g–i) used for the quantification of mitochondrial depolarisation following transfection with siNS (a and g), or siDAPK2 (b and h). Overall green fluorescence (FITC) data is also presented in histograms (U2OS: d and e; A549: j and k). Staining intensity was quantified using geometric means of three independent experiments and plotted as fold change (f and l). Data represent mean±S.E.M. of three independent experiments and the statistical analysis was done using Student's t-test (paired, one tailed) (*P<0.05). The phosphorylation of DAPK1 on Ser308 and DAPK1 expression levels were analysed in A549 lung cancer cells by SDS-PAGE/qWB, using HSP90 as a loading control (m)
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fig3: Genetic ablation of DAPK2 increases the rate of spontaneous mitochondrial membrane depolarisation using the JC-1 probe. U2OS (a–f) and A549 (g–m) cells were transfected with either siNS or siDAPK2. Forty-eight hours after transfection cells were incubated with JC-1 and the fluorescence of J-aggregates or monomers was measured in red and green fluorescence channels by flow cytometry. CCCP (50 μM) was used to induce complete mitochondrial depolarisation (c and i) and to set appropriate gates in U2OS (a–c) and A549 cells (g–i) used for the quantification of mitochondrial depolarisation following transfection with siNS (a and g), or siDAPK2 (b and h). Overall green fluorescence (FITC) data is also presented in histograms (U2OS: d and e; A549: j and k). Staining intensity was quantified using geometric means of three independent experiments and plotted as fold change (f and l). Data represent mean±S.E.M. of three independent experiments and the statistical analysis was done using Student's t-test (paired, one tailed) (*P<0.05). The phosphorylation of DAPK1 on Ser308 and DAPK1 expression levels were analysed in A549 lung cancer cells by SDS-PAGE/qWB, using HSP90 as a loading control (m)

Mentions: Elevated levels of mitochondrial O2•− can be both a cause and a consequence of mitochondrial depolarisation,23 which is involved in apoptosis and inflammation.24, 25 To study the effect of DAPK2 depletion on mitochondrial membrane potential (Δψm), the JC-1 probe was used. U2OS and A549 cells were transfected with either siNS, or siDAPK2 and, as a control for Δψm depolarisation, cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and then analysed by flow cytometry. Two distinct cell populations were identified after gating the depolarised population that resulted from CCCP treatment: one with intact mitochondria and another that harboured cells with depolarised mitochondria. Depletion of DAPK2 led to ~50% more depolarised mitochondria in both U2OS (Figures 3a–c) and A549 cells (Figures 3g–i), when compared with control cells. This pattern can also be seen in the histograms in Figures 3d, e, j and k. After normalising the absolute fluorescence of cells on DAPK2 knockdown to that of siNS-transfected cells, a significant overall increase in green fluorescence, a read-out for decreased Δψm, was measured in both cell lines (Figures 3f and l), suggesting that DAPK2 ablation increased spontaneous mitochondria depolarisation. This was confirmed using tetramethylrhodamine ethyl ester (TMRE), another mitochondrial probe (Figure 4).


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)

Genetic ablation of DAPK2 increases the rate of spontaneous mitochondrial membrane depolarisation using the JC-1 probe. U2OS (a–f) and A549 (g–m) cells were transfected with either siNS or siDAPK2. Forty-eight hours after transfection cells were incubated with JC-1 and the fluorescence of J-aggregates or monomers was measured in red and green fluorescence channels by flow cytometry. CCCP (50 μM) was used to induce complete mitochondrial depolarisation (c and i) and to set appropriate gates in U2OS (a–c) and A549 cells (g–i) used for the quantification of mitochondrial depolarisation following transfection with siNS (a and g), or siDAPK2 (b and h). Overall green fluorescence (FITC) data is also presented in histograms (U2OS: d and e; A549: j and k). Staining intensity was quantified using geometric means of three independent experiments and plotted as fold change (f and l). Data represent mean±S.E.M. of three independent experiments and the statistical analysis was done using Student's t-test (paired, one tailed) (*P<0.05). The phosphorylation of DAPK1 on Ser308 and DAPK1 expression levels were analysed in A549 lung cancer cells by SDS-PAGE/qWB, using HSP90 as a loading control (m)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Genetic ablation of DAPK2 increases the rate of spontaneous mitochondrial membrane depolarisation using the JC-1 probe. U2OS (a–f) and A549 (g–m) cells were transfected with either siNS or siDAPK2. Forty-eight hours after transfection cells were incubated with JC-1 and the fluorescence of J-aggregates or monomers was measured in red and green fluorescence channels by flow cytometry. CCCP (50 μM) was used to induce complete mitochondrial depolarisation (c and i) and to set appropriate gates in U2OS (a–c) and A549 cells (g–i) used for the quantification of mitochondrial depolarisation following transfection with siNS (a and g), or siDAPK2 (b and h). Overall green fluorescence (FITC) data is also presented in histograms (U2OS: d and e; A549: j and k). Staining intensity was quantified using geometric means of three independent experiments and plotted as fold change (f and l). Data represent mean±S.E.M. of three independent experiments and the statistical analysis was done using Student's t-test (paired, one tailed) (*P<0.05). The phosphorylation of DAPK1 on Ser308 and DAPK1 expression levels were analysed in A549 lung cancer cells by SDS-PAGE/qWB, using HSP90 as a loading control (m)
Mentions: Elevated levels of mitochondrial O2•− can be both a cause and a consequence of mitochondrial depolarisation,23 which is involved in apoptosis and inflammation.24, 25 To study the effect of DAPK2 depletion on mitochondrial membrane potential (Δψm), the JC-1 probe was used. U2OS and A549 cells were transfected with either siNS, or siDAPK2 and, as a control for Δψm depolarisation, cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and then analysed by flow cytometry. Two distinct cell populations were identified after gating the depolarised population that resulted from CCCP treatment: one with intact mitochondria and another that harboured cells with depolarised mitochondria. Depletion of DAPK2 led to ~50% more depolarised mitochondria in both U2OS (Figures 3a–c) and A549 cells (Figures 3g–i), when compared with control cells. This pattern can also be seen in the histograms in Figures 3d, e, j and k. After normalising the absolute fluorescence of cells on DAPK2 knockdown to that of siNS-transfected cells, a significant overall increase in green fluorescence, a read-out for decreased Δψm, was measured in both cell lines (Figures 3f and l), suggesting that DAPK2 ablation increased spontaneous mitochondria depolarisation. This was confirmed using tetramethylrhodamine ethyl ester (TMRE), another mitochondrial probe (Figure 4).

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