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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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Loss of Arg296 and Arg299 methylation on hnRNPK promotes the nearby Ser302 phosphorylation under etoposide treatment in vivo. (a) Ser302 phosphorylation of endogenous hnRNPK was up-regulated upon etoposide treatment. U2OS cells were treated with or without etoposide for 4 h. Endogenous hnRNPKs were immunoprecipitated from U2OS lysates, followed by western blot analysis to reveal the Ser302 phosphorylation of hnRNPK. N.S., non-specific. (b) Etoposide treatment reduced methylation level of endogenous hnRNPK. The U2OS cells were treated with or without etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection and expression of the precipitated hnRNPKs were carried out using pan-asymmetric methyl-arginine antibody and hnRNPK antibody, respectively. (c) Arg296 and Arg299 mutations increased the Ser302 phosphorylation of hnRNPK in U2OS cells treated with etoposide. U2OS cells were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation status. (d) hnRNPK methylation at Arg296 and Arg299, but not the other arginines, affects Ser302 phosphorylation. U2OS cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) or Flag-3RK-2 (R256K/R258K/R268K) hnRNPK, followed by etoposide treatment for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation state. (e) Mutations at Arg296 and Arg299 increased hnRNPK Ser302 phosphorylation in H1299 cells treated with etoposide. H1299 cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from H1299 lysates, followed by western blot analysis to reveal the hnRNPK Ser302 phosphorylation status.
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Figure 6: Loss of Arg296 and Arg299 methylation on hnRNPK promotes the nearby Ser302 phosphorylation under etoposide treatment in vivo. (a) Ser302 phosphorylation of endogenous hnRNPK was up-regulated upon etoposide treatment. U2OS cells were treated with or without etoposide for 4 h. Endogenous hnRNPKs were immunoprecipitated from U2OS lysates, followed by western blot analysis to reveal the Ser302 phosphorylation of hnRNPK. N.S., non-specific. (b) Etoposide treatment reduced methylation level of endogenous hnRNPK. The U2OS cells were treated with or without etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection and expression of the precipitated hnRNPKs were carried out using pan-asymmetric methyl-arginine antibody and hnRNPK antibody, respectively. (c) Arg296 and Arg299 mutations increased the Ser302 phosphorylation of hnRNPK in U2OS cells treated with etoposide. U2OS cells were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation status. (d) hnRNPK methylation at Arg296 and Arg299, but not the other arginines, affects Ser302 phosphorylation. U2OS cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) or Flag-3RK-2 (R256K/R258K/R268K) hnRNPK, followed by etoposide treatment for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation state. (e) Mutations at Arg296 and Arg299 increased hnRNPK Ser302 phosphorylation in H1299 cells treated with etoposide. H1299 cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from H1299 lysates, followed by western blot analysis to reveal the hnRNPK Ser302 phosphorylation status.

Mentions: PKCδ, a serine/threonine kinase, is involved in both intrinsic and extrinsic apoptotic pathways and is activated during apoptosis upon treatment with cytotoxic agents such as etoposide (45,46). In addition, PKCδ knockdown abolishes DNA damage-induced apoptosis in various cells (47–49). Thus, we further investigated whether etoposide treatment induces PKCδ-mediated phosphorylation of hnRNPK at Ser302. PKCδ is strongly activated after 4 h of etoposide treatment based on the maximal amount of PKCδ catalytic fragment (Supplementary Figure S2). Subsequently, hnRNPK was immunoprecipitated from U2OS cells with or without etoposide treatment, and Ser302 phosphorylation levels were determined using a phosphorylation-specific antibody. As shown in Figure 6a, the Ser302 phosphorylation of hnRNPK in etoposide-treated cells was higher than in untreated cells, suggesting that PKCδ-mediated phosphorylation is inducible through the etoposide treatment of U2OS cells. In addition, we further determined whether etoposide treatment affects the endogenous hnRNPK methylation status. The U2OS cells were treated with etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection of the precipitated hnRNPK was carried out using pan-asymmetric methyl-arginine antibody. As shown in Figure 6b, the methylation level of endogenous hnRNPK in U2OS cells was decreased after etoposide treatment, suggesting that the reduced methylation of hnRNPK is correlated with elevation of its Ser302 phosphorylation during etoposide treatment.


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)

Loss of Arg296 and Arg299 methylation on hnRNPK promotes the nearby Ser302 phosphorylation under etoposide treatment in vivo. (a) Ser302 phosphorylation of endogenous hnRNPK was up-regulated upon etoposide treatment. U2OS cells were treated with or without etoposide for 4 h. Endogenous hnRNPKs were immunoprecipitated from U2OS lysates, followed by western blot analysis to reveal the Ser302 phosphorylation of hnRNPK. N.S., non-specific. (b) Etoposide treatment reduced methylation level of endogenous hnRNPK. The U2OS cells were treated with or without etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection and expression of the precipitated hnRNPKs were carried out using pan-asymmetric methyl-arginine antibody and hnRNPK antibody, respectively. (c) Arg296 and Arg299 mutations increased the Ser302 phosphorylation of hnRNPK in U2OS cells treated with etoposide. U2OS cells were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation status. (d) hnRNPK methylation at Arg296 and Arg299, but not the other arginines, affects Ser302 phosphorylation. U2OS cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) or Flag-3RK-2 (R256K/R258K/R268K) hnRNPK, followed by etoposide treatment for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation state. (e) Mutations at Arg296 and Arg299 increased hnRNPK Ser302 phosphorylation in H1299 cells treated with etoposide. H1299 cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from H1299 lysates, followed by western blot analysis to reveal the hnRNPK Ser302 phosphorylation status.
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Figure 6: Loss of Arg296 and Arg299 methylation on hnRNPK promotes the nearby Ser302 phosphorylation under etoposide treatment in vivo. (a) Ser302 phosphorylation of endogenous hnRNPK was up-regulated upon etoposide treatment. U2OS cells were treated with or without etoposide for 4 h. Endogenous hnRNPKs were immunoprecipitated from U2OS lysates, followed by western blot analysis to reveal the Ser302 phosphorylation of hnRNPK. N.S., non-specific. (b) Etoposide treatment reduced methylation level of endogenous hnRNPK. The U2OS cells were treated with or without etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection and expression of the precipitated hnRNPKs were carried out using pan-asymmetric methyl-arginine antibody and hnRNPK antibody, respectively. (c) Arg296 and Arg299 mutations increased the Ser302 phosphorylation of hnRNPK in U2OS cells treated with etoposide. U2OS cells were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation status. (d) hnRNPK methylation at Arg296 and Arg299, but not the other arginines, affects Ser302 phosphorylation. U2OS cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) or Flag-3RK-2 (R256K/R258K/R268K) hnRNPK, followed by etoposide treatment for 4 h. The exogenous hnRNPKs were immunoprecipitated from U2OS lysates using a Flag antibody followed by western blot analysis to determine the hnRNPK Ser302 phosphorylation state. (e) Mutations at Arg296 and Arg299 increased hnRNPK Ser302 phosphorylation in H1299 cells treated with etoposide. H1299 cells carrying overexpressed Myc-tag PKCδ were transfected with Flag-WT or Flag-2RK (R296K/R299K) hnRNPK, followed by treatment with etoposide for 4 h. The exogenous hnRNPKs were immunoprecipitated from H1299 lysates, followed by western blot analysis to reveal the hnRNPK Ser302 phosphorylation status.
Mentions: PKCδ, a serine/threonine kinase, is involved in both intrinsic and extrinsic apoptotic pathways and is activated during apoptosis upon treatment with cytotoxic agents such as etoposide (45,46). In addition, PKCδ knockdown abolishes DNA damage-induced apoptosis in various cells (47–49). Thus, we further investigated whether etoposide treatment induces PKCδ-mediated phosphorylation of hnRNPK at Ser302. PKCδ is strongly activated after 4 h of etoposide treatment based on the maximal amount of PKCδ catalytic fragment (Supplementary Figure S2). Subsequently, hnRNPK was immunoprecipitated from U2OS cells with or without etoposide treatment, and Ser302 phosphorylation levels were determined using a phosphorylation-specific antibody. As shown in Figure 6a, the Ser302 phosphorylation of hnRNPK in etoposide-treated cells was higher than in untreated cells, suggesting that PKCδ-mediated phosphorylation is inducible through the etoposide treatment of U2OS cells. In addition, we further determined whether etoposide treatment affects the endogenous hnRNPK methylation status. The U2OS cells were treated with etoposide, and followed by immunoprecipitation of hnRNPK. Subsequent methylation detection of the precipitated hnRNPK was carried out using pan-asymmetric methyl-arginine antibody. As shown in Figure 6b, the methylation level of endogenous hnRNPK in U2OS cells was decreased after etoposide treatment, suggesting that the reduced methylation of hnRNPK is correlated with elevation of its Ser302 phosphorylation during etoposide treatment.

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