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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 preferentially methylates Arg296 and Arg299 of hnRNPK. (a) Detection and quantification of the recombinant PRMT1-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants, containing only one remaining arginine for methylation (as shown in the schematic diagram). HnRNPKs were incubated with GST-PRMT1 in the presence of [3H] S-adenosylmethionine (SAM), followed by SDS-PAGE. The proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants to WT hnRNPK are shown as a bar graph. (b) Detection and quantification of the PRMT1 complex-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants in vitro. HA-PRMT1 complex were immunoprecipitated from the HEK293 cells using an anti-HA antibody and incubated with diverse hnRNPKs in the presence of [3H] S-adenosylmethionine (SAM). After SDS-PAGE analysis, the proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants compared with WT hnRNPK are shown as a bar graph. CBB, Coomassie Brilliant Blue.
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Figure 2: PRMT1 preferentially methylates Arg296 and Arg299 of hnRNPK. (a) Detection and quantification of the recombinant PRMT1-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants, containing only one remaining arginine for methylation (as shown in the schematic diagram). HnRNPKs were incubated with GST-PRMT1 in the presence of [3H] S-adenosylmethionine (SAM), followed by SDS-PAGE. The proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants to WT hnRNPK are shown as a bar graph. (b) Detection and quantification of the PRMT1 complex-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants in vitro. HA-PRMT1 complex were immunoprecipitated from the HEK293 cells using an anti-HA antibody and incubated with diverse hnRNPKs in the presence of [3H] S-adenosylmethionine (SAM). After SDS-PAGE analysis, the proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants compared with WT hnRNPK are shown as a bar graph. CBB, Coomassie Brilliant Blue.

Mentions: PRMT1 catalyzes the transfer of methyl groups onto hnRNPK at five major sites including Arg256, Arg258, Arg268, Arg296 and Arg299 (28,29). Whether these different arginines exhibit similar efficiency and preference toward PRMT1-mediated methylation remains unknown. To this end, we established diverse hnRNPK arginine mutants exhibiting limited arginine methylation, as described in the ‘Materials and Methods’ section and Supplementary Table S1. Upon in vitro methylation, catalyzed through PRMT1 in the presence of [3H]-SAM, the 2RK, 3RK-1 and 5RK mutants all exhibited reduced methylation compared with WT hnRNPK (Figure 1a). Notably, the mutation of Arg296/Arg299 in 2RK hnRNPK resulted in a significant reduction of methylation, up to 60% of WT hnRNPK methylation. Next, we examined the in vivo methylation levels of WT, 2RK and 5RK hnRNPKs in HEK293T cells using an anti-methyl arginine antibody (7E6). As shown in Figure 1b, a similar loss of 60% WT methylation was observed for the Arg296/Arg299 mutation in 2RK hnRNPK. Accordingly, these results suggest that different arginines on hnRNPK are not chemically equivalent in PRMT1-mediated methylation. To explore this hypothesis, we further determined the efficiency and time dependence of the in vitro methylation on all five 4RK mutants containing only one arginine for methylation. The results showed that Arg299 and Arg296 exhibited the most and second-most efficient methylation, respectively, through either recombinant PRMT1 or the cellular PRMT1 complex (Figure 2a and b). In addition, time-dependent methylation of the five 4RK mutants revealed that Arg296 and Arg299 also had higher methylation rates than other arginines (Figure 3a and b). Taken together, these results demonstrated that the known methylable arginines in hnRNPK exhibit non-equivalent reactivity toward the PRMT1-catalyzed methylation. Moreover, Arg296 and Arg299 had higher methylation potential than other residues in vitro and in vivo, suggesting that these two residues might play distinct roles in the regulation of hnRNPK functions.


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 preferentially methylates Arg296 and Arg299 of hnRNPK. (a) Detection and quantification of the recombinant PRMT1-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants, containing only one remaining arginine for methylation (as shown in the schematic diagram). HnRNPKs were incubated with GST-PRMT1 in the presence of [3H] S-adenosylmethionine (SAM), followed by SDS-PAGE. The proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants to WT hnRNPK are shown as a bar graph. (b) Detection and quantification of the PRMT1 complex-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants in vitro. HA-PRMT1 complex were immunoprecipitated from the HEK293 cells using an anti-HA antibody and incubated with diverse hnRNPKs in the presence of [3H] S-adenosylmethionine (SAM). After SDS-PAGE analysis, the proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants compared with WT hnRNPK are shown as a bar graph. CBB, Coomassie Brilliant Blue.
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Related In: Results  -  Collection

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Figure 2: PRMT1 preferentially methylates Arg296 and Arg299 of hnRNPK. (a) Detection and quantification of the recombinant PRMT1-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants, containing only one remaining arginine for methylation (as shown in the schematic diagram). HnRNPKs were incubated with GST-PRMT1 in the presence of [3H] S-adenosylmethionine (SAM), followed by SDS-PAGE. The proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants to WT hnRNPK are shown as a bar graph. (b) Detection and quantification of the PRMT1 complex-mediated methylation of WT hnRNPK, the 5RK mutant and five 4RK mutants in vitro. HA-PRMT1 complex were immunoprecipitated from the HEK293 cells using an anti-HA antibody and incubated with diverse hnRNPKs in the presence of [3H] S-adenosylmethionine (SAM). After SDS-PAGE analysis, the proteins were stained with Coomassie blue (bottom). Methylation was detected through fluorography (top) and quantified using a liquid scintillation counter. The relative methylation levels of all mutants compared with WT hnRNPK are shown as a bar graph. CBB, Coomassie Brilliant Blue.
Mentions: PRMT1 catalyzes the transfer of methyl groups onto hnRNPK at five major sites including Arg256, Arg258, Arg268, Arg296 and Arg299 (28,29). Whether these different arginines exhibit similar efficiency and preference toward PRMT1-mediated methylation remains unknown. To this end, we established diverse hnRNPK arginine mutants exhibiting limited arginine methylation, as described in the ‘Materials and Methods’ section and Supplementary Table S1. Upon in vitro methylation, catalyzed through PRMT1 in the presence of [3H]-SAM, the 2RK, 3RK-1 and 5RK mutants all exhibited reduced methylation compared with WT hnRNPK (Figure 1a). Notably, the mutation of Arg296/Arg299 in 2RK hnRNPK resulted in a significant reduction of methylation, up to 60% of WT hnRNPK methylation. Next, we examined the in vivo methylation levels of WT, 2RK and 5RK hnRNPKs in HEK293T cells using an anti-methyl arginine antibody (7E6). As shown in Figure 1b, a similar loss of 60% WT methylation was observed for the Arg296/Arg299 mutation in 2RK hnRNPK. Accordingly, these results suggest that different arginines on hnRNPK are not chemically equivalent in PRMT1-mediated methylation. To explore this hypothesis, we further determined the efficiency and time dependence of the in vitro methylation on all five 4RK mutants containing only one arginine for methylation. The results showed that Arg299 and Arg296 exhibited the most and second-most efficient methylation, respectively, through either recombinant PRMT1 or the cellular PRMT1 complex (Figure 2a and b). In addition, time-dependent methylation of the five 4RK mutants revealed that Arg296 and Arg299 also had higher methylation rates than other arginines (Figure 3a and b). Taken together, these results demonstrated that the known methylable arginines in hnRNPK exhibit non-equivalent reactivity toward the PRMT1-catalyzed methylation. Moreover, Arg296 and Arg299 had higher methylation potential than other residues in vitro and in vivo, suggesting that these two residues might play distinct roles in the regulation of hnRNPK functions.

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