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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.

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Blockage of Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. (a) SaOS-2 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 was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. (b) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.
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Figure 9: Blockage of Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. (a) SaOS-2 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 was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. (b) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.

Mentions: hnRNPK physically interacts with p53 as a co-transcriptional activator to regulate the downstream genes responsible for cell cycle arrest and apoptosis (27). However, co-immunoprecipitation experiments showed that the interaction of p53 and hnRNPK is not affected by the methylation defects of hnRNPK in U2OS cells after etoposide treatment and PKCδ overexpression (Supplementary Figure S8). Notably, p53-independent hnRNPK-mediated apoptosis has recently been reported (21). To determine whether the observed apoptotic induction through methylation-defective hnRNPK is p53 independent, we further established p53- SaOS2 cells carrying WT and 2RK hnRNPKs, namely SaOS2-K-WT and SaOS2-K-2RK cells (Figure 9a). hnRNPK immunoprecipitation experiments in SaOS2-K-WT and SaOS2-K-2RK cells treated with etoposide and PKCδ overexpression showed that 2RK hnRNPK exhibited higher Ser302 phosphorylation than WT hnRNPK, as shown in Figure 9b. Further analysis showed that, after a 12-h etoposide treatment, the SaOS2-K-2RK cells exhibited a higher degree of caspase 3 cleavage than SaOS2-K-WT cells. In addition, PKCδ overexpression and etoposide treatment together in both cells further increased the differences in caspase 3 cleavage between SaOS2-K-WT and SaOS2-K-2RK cells (Figure 9b). Taken together, these results suggest that the methylation defect in hnRNPK promotes caspase 3 activity and cellular apoptosis during etoposide-induced DNA damage in a p53-independent manner.


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 Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. (a) SaOS-2 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 was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. (b) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.
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Related In: Results  -  Collection

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Figure 9: Blockage of Arg296 and Arg299 methylation in hnRNPK promotes apoptosis in a p53-independent manner. (a) SaOS-2 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 was determined after measuring the protein levels of endogenous and exogenous hnRNPKs using hnRNPK and GAPDH antibodies. (b) The SaOS2-K-WT and SaOS2-K-2RK (R296K/R299K) cells were transfected with Myc-PKCδ for 24 h and subsequently treated with etoposide for 12 h. The cell lysates were collected and analyzed for Myc-PKCδ, GAPDH and cleaved caspase 3 expression levels using specific antibodies.
Mentions: hnRNPK physically interacts with p53 as a co-transcriptional activator to regulate the downstream genes responsible for cell cycle arrest and apoptosis (27). However, co-immunoprecipitation experiments showed that the interaction of p53 and hnRNPK is not affected by the methylation defects of hnRNPK in U2OS cells after etoposide treatment and PKCδ overexpression (Supplementary Figure S8). Notably, p53-independent hnRNPK-mediated apoptosis has recently been reported (21). To determine whether the observed apoptotic induction through methylation-defective hnRNPK is p53 independent, we further established p53- SaOS2 cells carrying WT and 2RK hnRNPKs, namely SaOS2-K-WT and SaOS2-K-2RK cells (Figure 9a). hnRNPK immunoprecipitation experiments in SaOS2-K-WT and SaOS2-K-2RK cells treated with etoposide and PKCδ overexpression showed that 2RK hnRNPK exhibited higher Ser302 phosphorylation than WT hnRNPK, as shown in Figure 9b. Further analysis showed that, after a 12-h etoposide treatment, the SaOS2-K-2RK cells exhibited a higher degree of caspase 3 cleavage than SaOS2-K-WT cells. In addition, PKCδ overexpression and etoposide treatment together in both cells further increased the differences in caspase 3 cleavage between SaOS2-K-WT and SaOS2-K-2RK cells (Figure 9b). Taken together, these results suggest that the methylation defect in hnRNPK promotes caspase 3 activity and cellular apoptosis during etoposide-induced DNA damage in a p53-independent manner.

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