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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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PRMT1-mediated methylation of hnRNPK suppresses its own PKCδ-mediated phosphorylation in vitro. (a) WT (GST-K) and pre-methylated hnRNPK (Me-GST-K) were purified from Escherichia coli, as described in the ‘Materials and Methods’ section. The methylation levels of both hnRNPKs were determined using an asymmetric dimethylarginine-specific antibody (7E6). (b) In vitro phosphorylation of GST-K and Me-GST-K. GST-K and Me-GST-K were incubated with the GST-catalytic fragment PKCδ GST-CF-PKCδ in the presence of [32P]-ATP, followed by SDS-PAGE analysis. The proteins were stained with Coomassie blue (right). The phosphorylation levels were analyzed through autoradiography (left). The relative phosphorylation levels of Me-GST-K to GST-K were quantified using ImageJ software (NIH) and shown as a bar graph. Single asterisk indicates statistical significance (p < 0.05).
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Figure 5: PRMT1-mediated methylation of hnRNPK suppresses its own PKCδ-mediated phosphorylation in vitro. (a) WT (GST-K) and pre-methylated hnRNPK (Me-GST-K) were purified from Escherichia coli, as described in the ‘Materials and Methods’ section. The methylation levels of both hnRNPKs were determined using an asymmetric dimethylarginine-specific antibody (7E6). (b) In vitro phosphorylation of GST-K and Me-GST-K. GST-K and Me-GST-K were incubated with the GST-catalytic fragment PKCδ GST-CF-PKCδ in the presence of [32P]-ATP, followed by SDS-PAGE analysis. The proteins were stained with Coomassie blue (right). The phosphorylation levels were analyzed through autoradiography (left). The relative phosphorylation levels of Me-GST-K to GST-K were quantified using ImageJ software (NIH) and shown as a bar graph. Single asterisk indicates statistical significance (p < 0.05).

Mentions: We next investigated whether arginine methylation of hnRNPK interferes with Ser302 phosphorylation. To this end, we generated M-GST-K, a recombinant GST-hnRNPK fusion protein carrying pre-methylated arginines, in E. coli as described in the ‘Materials and Methods’ section. The pre-methylation of this M-GST-K was verified using a methyl arginine-specific antibody, and the control GST-K showed no methylation signal (Figure 5a). Next, we performed the in vitro phosphorylation of pre-methylated M-GST-K and the un-methylated control (GST-K) using constitutively active PKCδ (catalytic fragment, CF-PKCδ) in the presence of [γ-32P]-ATP. A comparison of the phosphorylation levels showed that GST-K exhibited higher phosphorylation levels than M-GST-K (Figure 5b), suggesting that the pre-methylation of hnRNPK suppressed subsequent PKCδ-mediated phosphorylation in vitro.


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)

PRMT1-mediated methylation of hnRNPK suppresses its own PKCδ-mediated phosphorylation in vitro. (a) WT (GST-K) and pre-methylated hnRNPK (Me-GST-K) were purified from Escherichia coli, as described in the ‘Materials and Methods’ section. The methylation levels of both hnRNPKs were determined using an asymmetric dimethylarginine-specific antibody (7E6). (b) In vitro phosphorylation of GST-K and Me-GST-K. GST-K and Me-GST-K were incubated with the GST-catalytic fragment PKCδ GST-CF-PKCδ in the presence of [32P]-ATP, followed by SDS-PAGE analysis. The proteins were stained with Coomassie blue (right). The phosphorylation levels were analyzed through autoradiography (left). The relative phosphorylation levels of Me-GST-K to GST-K were quantified using ImageJ software (NIH) and shown as a bar graph. Single asterisk indicates statistical significance (p < 0.05).
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Figure 5: PRMT1-mediated methylation of hnRNPK suppresses its own PKCδ-mediated phosphorylation in vitro. (a) WT (GST-K) and pre-methylated hnRNPK (Me-GST-K) were purified from Escherichia coli, as described in the ‘Materials and Methods’ section. The methylation levels of both hnRNPKs were determined using an asymmetric dimethylarginine-specific antibody (7E6). (b) In vitro phosphorylation of GST-K and Me-GST-K. GST-K and Me-GST-K were incubated with the GST-catalytic fragment PKCδ GST-CF-PKCδ in the presence of [32P]-ATP, followed by SDS-PAGE analysis. The proteins were stained with Coomassie blue (right). The phosphorylation levels were analyzed through autoradiography (left). The relative phosphorylation levels of Me-GST-K to GST-K were quantified using ImageJ software (NIH) and shown as a bar graph. Single asterisk indicates statistical significance (p < 0.05).
Mentions: We next investigated whether arginine methylation of hnRNPK interferes with Ser302 phosphorylation. To this end, we generated M-GST-K, a recombinant GST-hnRNPK fusion protein carrying pre-methylated arginines, in E. coli as described in the ‘Materials and Methods’ section. The pre-methylation of this M-GST-K was verified using a methyl arginine-specific antibody, and the control GST-K showed no methylation signal (Figure 5a). Next, we performed the in vitro phosphorylation of pre-methylated M-GST-K and the un-methylated control (GST-K) using constitutively active PKCδ (catalytic fragment, CF-PKCδ) in the presence of [γ-32P]-ATP. A comparison of the phosphorylation levels showed that GST-K exhibited higher phosphorylation levels than M-GST-K (Figure 5b), suggesting that the pre-methylation of hnRNPK suppressed subsequent PKCδ-mediated phosphorylation in vitro.

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