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Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA.

Bikkavilli RK, Malbon CC - J. Cell. Sci. (2011)

Bottom Line: Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1.Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a.Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. kamesh@pharm.stonybrook.edu

ABSTRACT
Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

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Arginine methylation of G3BP1 impairs its binding to Ctnnb1 mRNA and Dvl3. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, wild-type G3BP1, its methylation-deficient mutants (433K, 445K) or its methylation-mimicking mutants (433F, 445F) with anti-Myc antibodies. The amount of Ctnnb1 mRNA in the immunoprecipitates was then quantified using quantitative PCR. **P<0.01 versus control (pCMV-Myc). (B) Northwestern analysis of unmethylated or methylated GST–G3BP1 and Ctnnb1 mRNA. Equal amounts of unmethylated or methylated (with PRMT1 isolated from unstimulated cells or cells treated with Wnt3a for 6 hours and [3H]SAM) GST-G3BP1 were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed on the blots using DIG-labeled Ctnnb1 UTR. The binding of Ctnnb1 mRNA to unmethylated GST–G3BP1 was taken as 100%. The top panel represents mean values ± s.e.m. obtained from three independent experiments; the bottom panels display northwestern blots and the corresponding fluorograph. *P<0.05; **P<0.01 versus control (unmethylated GST–G3BP1). (C) F9 cells were transiently transfected with either pCMV–Myc, Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies. Interaction of G3BP1 and its mutants with Dvl3 was made visible by probing the blots with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent experiments; bottom panel displays representative blots. **P<0.01 versus control (WT). (D) F9 cells were transiently transfected with methylation-deficient mutants of Myc–G3BP1 (R433K, R445K) for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for indicated periods of time followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies followed by immunoblotting with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus corresponding unstimulated control (–Wnt3a). (E) F9 cells were transfected either with Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription following stimulation with Wnt3a for 7 hours. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; bottom panel displays representative blots. ##P<0.01 versus control (pCMV-Myc).
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Figure 6: Arginine methylation of G3BP1 impairs its binding to Ctnnb1 mRNA and Dvl3. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, wild-type G3BP1, its methylation-deficient mutants (433K, 445K) or its methylation-mimicking mutants (433F, 445F) with anti-Myc antibodies. The amount of Ctnnb1 mRNA in the immunoprecipitates was then quantified using quantitative PCR. **P<0.01 versus control (pCMV-Myc). (B) Northwestern analysis of unmethylated or methylated GST–G3BP1 and Ctnnb1 mRNA. Equal amounts of unmethylated or methylated (with PRMT1 isolated from unstimulated cells or cells treated with Wnt3a for 6 hours and [3H]SAM) GST-G3BP1 were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed on the blots using DIG-labeled Ctnnb1 UTR. The binding of Ctnnb1 mRNA to unmethylated GST–G3BP1 was taken as 100%. The top panel represents mean values ± s.e.m. obtained from three independent experiments; the bottom panels display northwestern blots and the corresponding fluorograph. *P<0.05; **P<0.01 versus control (unmethylated GST–G3BP1). (C) F9 cells were transiently transfected with either pCMV–Myc, Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies. Interaction of G3BP1 and its mutants with Dvl3 was made visible by probing the blots with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent experiments; bottom panel displays representative blots. **P<0.01 versus control (WT). (D) F9 cells were transiently transfected with methylation-deficient mutants of Myc–G3BP1 (R433K, R445K) for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for indicated periods of time followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies followed by immunoblotting with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus corresponding unstimulated control (–Wnt3a). (E) F9 cells were transfected either with Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription following stimulation with Wnt3a for 7 hours. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; bottom panel displays representative blots. ##P<0.01 versus control (pCMV-Myc).

Mentions: Does methylation of G3BP1 alter its binding of Ctnnb1 mRNA? Myc-tagged wild-type G3BP1 and methylation-deficient (R433K, R445K) and methylation-mimicking [R433F, R445F (Mostaqul Huq et al., 2006; Weber et al., 2009; Guo et al., 2010)] mutants of G3BP1 were used to address this question. Cells were transiently transfected with either wild-type or mutant forms of G3BP1 and cell lysates were later subjected to pull-downs with anti-Myc antibodies. Isolation of RNA (from Myc pull-downs) and amplification by RT-PCR was performed next. The relative amounts of Ctnnb1 transcripts in the G3BP1 complexes were then established using quantitative real-time PCR. Ctnnb1 transcripts were identified in the wild-type G3BP1 pull-downs (Fig. 6A). By contrast, Ctnnb1 transcripts were nearly undetectable in the pull-downs from cells expressing R433F mutant of G3BP1 (a ‘methylation-mimicking mutant’, Fig. 6A). Binding of Ctnnb1 mRNA to G3BP1 was unaffected by the R445F mutation, being similar to either wild-type or methylation-deficient mutants (R433K, R445K, Fig. 6A). To further test the role of arginine methylation in the association of G3BP1 with Ctnnb1 mRNA, we examined whether methylation of GST–G3BP1 affects its ability to bind Ctnnb1 mRNA in vitro. For these experiments, methylated GST–G3BP1 was prepared in vitro using HA–PRMT1. Methylation was assessed through the use of tritiated-S-adenosyl methionine ([3H]SAM) in the methylation assay buffer. Equal amounts of unmethylated and methylated GST–G3BP1 were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Northwestern analysis was then performed on the membranes using a DIG-labeled full-length Ctnnb1 UTR. Consistent with the RNA immunoprecipitation data (Fig. 6A), Wnt3a-stimulated PRMT1-mediated methylation of GST–G3BP1 provoked a sharp decrease in the ability of G3BP1 to bind Ctnnb1 mRNA (Fig. 6B). Methylation of GST–G3BP1 by PRMT1 isolated from untreated cells provoked a small decrease in the ability of G3BP1 to bind Ctnnb1 mRNA (Fig. 6B). Arginine methylation of G3BP1 appears to be a molecular switch: in response to methylation at R433, the ability of G3BP1 to bind Ctnnb1 mRNA was sharply attenuated (Fig. 6A,B).


Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA.

Bikkavilli RK, Malbon CC - J. Cell. Sci. (2011)

Arginine methylation of G3BP1 impairs its binding to Ctnnb1 mRNA and Dvl3. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, wild-type G3BP1, its methylation-deficient mutants (433K, 445K) or its methylation-mimicking mutants (433F, 445F) with anti-Myc antibodies. The amount of Ctnnb1 mRNA in the immunoprecipitates was then quantified using quantitative PCR. **P<0.01 versus control (pCMV-Myc). (B) Northwestern analysis of unmethylated or methylated GST–G3BP1 and Ctnnb1 mRNA. Equal amounts of unmethylated or methylated (with PRMT1 isolated from unstimulated cells or cells treated with Wnt3a for 6 hours and [3H]SAM) GST-G3BP1 were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed on the blots using DIG-labeled Ctnnb1 UTR. The binding of Ctnnb1 mRNA to unmethylated GST–G3BP1 was taken as 100%. The top panel represents mean values ± s.e.m. obtained from three independent experiments; the bottom panels display northwestern blots and the corresponding fluorograph. *P<0.05; **P<0.01 versus control (unmethylated GST–G3BP1). (C) F9 cells were transiently transfected with either pCMV–Myc, Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies. Interaction of G3BP1 and its mutants with Dvl3 was made visible by probing the blots with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent experiments; bottom panel displays representative blots. **P<0.01 versus control (WT). (D) F9 cells were transiently transfected with methylation-deficient mutants of Myc–G3BP1 (R433K, R445K) for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for indicated periods of time followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies followed by immunoblotting with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus corresponding unstimulated control (–Wnt3a). (E) F9 cells were transfected either with Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription following stimulation with Wnt3a for 7 hours. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; bottom panel displays representative blots. ##P<0.01 versus control (pCMV-Myc).
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Figure 6: Arginine methylation of G3BP1 impairs its binding to Ctnnb1 mRNA and Dvl3. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, wild-type G3BP1, its methylation-deficient mutants (433K, 445K) or its methylation-mimicking mutants (433F, 445F) with anti-Myc antibodies. The amount of Ctnnb1 mRNA in the immunoprecipitates was then quantified using quantitative PCR. **P<0.01 versus control (pCMV-Myc). (B) Northwestern analysis of unmethylated or methylated GST–G3BP1 and Ctnnb1 mRNA. Equal amounts of unmethylated or methylated (with PRMT1 isolated from unstimulated cells or cells treated with Wnt3a for 6 hours and [3H]SAM) GST-G3BP1 were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed on the blots using DIG-labeled Ctnnb1 UTR. The binding of Ctnnb1 mRNA to unmethylated GST–G3BP1 was taken as 100%. The top panel represents mean values ± s.e.m. obtained from three independent experiments; the bottom panels display northwestern blots and the corresponding fluorograph. *P<0.05; **P<0.01 versus control (unmethylated GST–G3BP1). (C) F9 cells were transiently transfected with either pCMV–Myc, Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies. Interaction of G3BP1 and its mutants with Dvl3 was made visible by probing the blots with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent experiments; bottom panel displays representative blots. **P<0.01 versus control (WT). (D) F9 cells were transiently transfected with methylation-deficient mutants of Myc–G3BP1 (R433K, R445K) for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for indicated periods of time followed by cell lysis and affinity pull-downs with anti-Dvl3 antibodies followed by immunoblotting with anti-Myc antibodies. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus corresponding unstimulated control (–Wnt3a). (E) F9 cells were transfected either with Myc–G3BP1 or its methylation-deficient mutants (R433K, R445K) for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription following stimulation with Wnt3a for 7 hours. Top panel displays mean values ± s.e.m. obtained from three independent highly reproducible experiments; bottom panel displays representative blots. ##P<0.01 versus control (pCMV-Myc).
Mentions: Does methylation of G3BP1 alter its binding of Ctnnb1 mRNA? Myc-tagged wild-type G3BP1 and methylation-deficient (R433K, R445K) and methylation-mimicking [R433F, R445F (Mostaqul Huq et al., 2006; Weber et al., 2009; Guo et al., 2010)] mutants of G3BP1 were used to address this question. Cells were transiently transfected with either wild-type or mutant forms of G3BP1 and cell lysates were later subjected to pull-downs with anti-Myc antibodies. Isolation of RNA (from Myc pull-downs) and amplification by RT-PCR was performed next. The relative amounts of Ctnnb1 transcripts in the G3BP1 complexes were then established using quantitative real-time PCR. Ctnnb1 transcripts were identified in the wild-type G3BP1 pull-downs (Fig. 6A). By contrast, Ctnnb1 transcripts were nearly undetectable in the pull-downs from cells expressing R433F mutant of G3BP1 (a ‘methylation-mimicking mutant’, Fig. 6A). Binding of Ctnnb1 mRNA to G3BP1 was unaffected by the R445F mutation, being similar to either wild-type or methylation-deficient mutants (R433K, R445K, Fig. 6A). To further test the role of arginine methylation in the association of G3BP1 with Ctnnb1 mRNA, we examined whether methylation of GST–G3BP1 affects its ability to bind Ctnnb1 mRNA in vitro. For these experiments, methylated GST–G3BP1 was prepared in vitro using HA–PRMT1. Methylation was assessed through the use of tritiated-S-adenosyl methionine ([3H]SAM) in the methylation assay buffer. Equal amounts of unmethylated and methylated GST–G3BP1 were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Northwestern analysis was then performed on the membranes using a DIG-labeled full-length Ctnnb1 UTR. Consistent with the RNA immunoprecipitation data (Fig. 6A), Wnt3a-stimulated PRMT1-mediated methylation of GST–G3BP1 provoked a sharp decrease in the ability of G3BP1 to bind Ctnnb1 mRNA (Fig. 6B). Methylation of GST–G3BP1 by PRMT1 isolated from untreated cells provoked a small decrease in the ability of G3BP1 to bind Ctnnb1 mRNA (Fig. 6B). Arginine methylation of G3BP1 appears to be a molecular switch: in response to methylation at R433, the ability of G3BP1 to bind Ctnnb1 mRNA was sharply attenuated (Fig. 6A,B).

Bottom Line: Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1.Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a.Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. kamesh@pharm.stonybrook.edu

ABSTRACT
Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

Show MeSH
Related in: MedlinePlus