Limits...
Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation.

Yang JH, Chiou YY, Fu SL, Shih IY, Weng TH, Lin WJ, Lin CH - Nucleic Acids Res. (2014)

Bottom Line: In addition, increased hnRNPK expression has been associated with tumor development and progression.In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ.While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang Ming University, Taipei 11221, Taiwan.

Show MeSH

Related in: MedlinePlus

Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. (a) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. (b) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. (c) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. (d) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. (e) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150800&req=5

Figure 7: Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. (a) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. (b) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. (c) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. (d) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. (e) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.

Mentions: HnRNPK is highly abundant in cells, and its gene knockout is lethal in several species (1,50–52). In addition, the aggressive knockdown of endogenous hnRNPK in cells leads to cell death (12,21–23). To investigate the putative function of Arg296/Arg299 methylation of hnRNPK in vivo, we first overexpressed exogenous hnRNPK carrying diverse arginine mutations in U2OS cells. However, an insignificant phenotype was observed, likely reflecting the abundant presence of endogenous hnRNPK. We thus further established stable cell lines expressing exogenous WT hnRNPK or hnRNPK-2RK (R296K/R299K) and knocked down endogenous hnRNPK through lentivirus-based RNA interference, as described in the ‘Materials and Methods’ section. The resulting cells, U2OS-K-WT and U2OS-K-2RK cells, expressed a significant amount of exogenous hnRNPK-WT or hnRNPK-2RK over the endogenous hnRNPK (Figure 7a). Despite the arginine mutations in hnRNPK, the growth rate and morphology of these two cell lines were nearly identical (data not shown).


Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation.

Yang JH, Chiou YY, Fu SL, Shih IY, Weng TH, Lin WJ, Lin CH - Nucleic Acids Res. (2014)

Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. (a) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. (b) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. (c) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. (d) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. (e) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150800&req=5

Figure 7: Blockage of Arg299 and Arg296 methylation in hnRNPK promotes U2OS cell apoptosis upon DNA damage. (a) Establishment of stable cell lines carrying Arg296 and Arg299 methylation-defective hnRNPK. U2OS cells were simultaneously infected with lentivirus carrying shRNA against endogenous hnRNPK and lentivirus carrying shRNA-resistant WT or 2RK mutant hnRNPKs. The efficiency of knockdown and ectopic expression were determined according to the protein levels of endogenous and exogenous hnRNPKs, measured using hnRNPK and GAPDH antibodies. (b) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and treated with etoposide for the indicated times. The cell lysates were collected, stained with propidium iodide and measured through FACS to calculate the percentages of sub-G1 cells. The data are shown as the mean value and SD from three independent experiments. (c) Under the same treatment as described above, the cells were collected at the indicated times and analyzed using a TUNEL assay to determine the percentages of apoptotic cells through FACS. (d) U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells under the same treatment at 12 h were collected and analyzed for the expression levels of Myc-PKCδ, GAPDH and cleaved caspase 3 using specific antibodies. Pretreatment with the PKCδ inhibitor rottlerin in U2OS-K-2RK cells prior to etoposide treatment was also performed. (e) Arg296 and Arg299 methylation-defective hnRNPK promotes apoptosis via both extrinsic and intrinsic pathways. U2OS-K-WT and U2OS-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ, followed by etoposide treatment for 12 h. The expression levels of Myc-PKCδ and GAPDH and cleaved caspases 3, 8 and 9 were measured using specific antibodies.
Mentions: HnRNPK is highly abundant in cells, and its gene knockout is lethal in several species (1,50–52). In addition, the aggressive knockdown of endogenous hnRNPK in cells leads to cell death (12,21–23). To investigate the putative function of Arg296/Arg299 methylation of hnRNPK in vivo, we first overexpressed exogenous hnRNPK carrying diverse arginine mutations in U2OS cells. However, an insignificant phenotype was observed, likely reflecting the abundant presence of endogenous hnRNPK. We thus further established stable cell lines expressing exogenous WT hnRNPK or hnRNPK-2RK (R296K/R299K) and knocked down endogenous hnRNPK through lentivirus-based RNA interference, as described in the ‘Materials and Methods’ section. The resulting cells, U2OS-K-WT and U2OS-K-2RK cells, expressed a significant amount of exogenous hnRNPK-WT or hnRNPK-2RK over the endogenous hnRNPK (Figure 7a). Despite the arginine mutations in hnRNPK, the growth rate and morphology of these two cell lines were nearly identical (data not shown).

Bottom Line: In addition, increased hnRNPK expression has been associated with tumor development and progression.In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ.While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang Ming University, Taipei 11221, Taiwan.

Show MeSH
Related in: MedlinePlus