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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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PRMT1 binds to and methylates G3BP1. (A) Ectopic PRMT1 interacts with G3BP1. F9 cells were transiently transfected with Myc–G3BP1 either alone or with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with anti-HA antibodies. Interaction of G3BP1 with PRMT1 was visualized by probing the blots with anti-Myc antibodies. F9 cells were transiently trasfected with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with control IgG, anti-Dvl3, anti-GSK3β or anti-HA antibodies. The association of PRMT1 with different proteins was visualized by probing the blots with anti-HA antibodies. (B) F9 cells were transiently transfected with HA–PRMT1 and Myc–G3BP1 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-HA specific 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. Asterisks indicate the bands of Immunoglobulin heavy chain. Representative blots of three independent experiments that proved highly reproducible were displayed. *P<0.05 versus unstimulated control (–Wnt3a). (C) In vitro methylation assay for G3BP1. F9 cells were transiently transfected with empty vector or HA–PRMT1 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-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 as substrate and [3H]SAM as a methyl donor. Top panel displays the d.p.m. of the incorporated tritiated methyl groups measured from the excised bands of the SDS-PAGE gel and the bottom panel displays a representative fluorograph. (D) In vitro methylation assay for G3BP1 mutants. F9 cells were transiently transfected with HA–PRMT1 for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for 6 h followed by cell lysis and affinity pull-downs with anti-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 and its mutants R433K or R445K as substrates and [3H]SAM as a methyl donor. Representative data of two independent experiments that proved highly reproducible were displayed. (E) In vivo methylation of G3BP1. F9 cells were transiently transfected with empty vector, Myc–G3BP1, or G3BP1 mutants for 24 hours. The cells were then metabolically labeled with L-[methyl-3H]methionine in the presence of protein translation inhibitors. After 3 hours, the cells were lysed and affinity pull-downs with anti-Myc antibodies were performed. The methylation statuses of G3BP1 and its mutants were then revealed by SDS-PAGE followed by fluorography. After fluorography, the gels were rehydrated, transferred to nitrocellulose membranes followed by immunoblotting with anti-Myc antibodies (bottom panel). Representative data of two independent experiments that proved highly reproducible were displayed.
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Figure 3: PRMT1 binds to and methylates G3BP1. (A) Ectopic PRMT1 interacts with G3BP1. F9 cells were transiently transfected with Myc–G3BP1 either alone or with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with anti-HA antibodies. Interaction of G3BP1 with PRMT1 was visualized by probing the blots with anti-Myc antibodies. F9 cells were transiently trasfected with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with control IgG, anti-Dvl3, anti-GSK3β or anti-HA antibodies. The association of PRMT1 with different proteins was visualized by probing the blots with anti-HA antibodies. (B) F9 cells were transiently transfected with HA–PRMT1 and Myc–G3BP1 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-HA specific 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. Asterisks indicate the bands of Immunoglobulin heavy chain. Representative blots of three independent experiments that proved highly reproducible were displayed. *P<0.05 versus unstimulated control (–Wnt3a). (C) In vitro methylation assay for G3BP1. F9 cells were transiently transfected with empty vector or HA–PRMT1 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-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 as substrate and [3H]SAM as a methyl donor. Top panel displays the d.p.m. of the incorporated tritiated methyl groups measured from the excised bands of the SDS-PAGE gel and the bottom panel displays a representative fluorograph. (D) In vitro methylation assay for G3BP1 mutants. F9 cells were transiently transfected with HA–PRMT1 for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for 6 h followed by cell lysis and affinity pull-downs with anti-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 and its mutants R433K or R445K as substrates and [3H]SAM as a methyl donor. Representative data of two independent experiments that proved highly reproducible were displayed. (E) In vivo methylation of G3BP1. F9 cells were transiently transfected with empty vector, Myc–G3BP1, or G3BP1 mutants for 24 hours. The cells were then metabolically labeled with L-[methyl-3H]methionine in the presence of protein translation inhibitors. After 3 hours, the cells were lysed and affinity pull-downs with anti-Myc antibodies were performed. The methylation statuses of G3BP1 and its mutants were then revealed by SDS-PAGE followed by fluorography. After fluorography, the gels were rehydrated, transferred to nitrocellulose membranes followed by immunoblotting with anti-Myc antibodies (bottom panel). Representative data of two independent experiments that proved highly reproducible were displayed.

Mentions: Arginine methylation of proteins can be catalyzed by protein arginine methyl transferase 1 (PRMT1), a ubiquitously expressed methyl transferase for histones and other nuclear proteins that bind nucleic acids (Bedford and Clarke, 2009; Lee and Stallcup, 2009). Because G3BP1 was prominently methylated by Wnt3a in the Dvl3-based complex, we evaluated whether PRMT1 was catalyzing the process. Pull-downs performed on F9 cell extracts expressing HA-tagged PRMT1 and Myc-tagged G3BP1 revealed PRMT1–G3BP1 association in these complexes (Fig. 3A). Pull-downsFig. 1.


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

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

PRMT1 binds to and methylates G3BP1. (A) Ectopic PRMT1 interacts with G3BP1. F9 cells were transiently transfected with Myc–G3BP1 either alone or with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with anti-HA antibodies. Interaction of G3BP1 with PRMT1 was visualized by probing the blots with anti-Myc antibodies. F9 cells were transiently trasfected with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with control IgG, anti-Dvl3, anti-GSK3β or anti-HA antibodies. The association of PRMT1 with different proteins was visualized by probing the blots with anti-HA antibodies. (B) F9 cells were transiently transfected with HA–PRMT1 and Myc–G3BP1 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-HA specific 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. Asterisks indicate the bands of Immunoglobulin heavy chain. Representative blots of three independent experiments that proved highly reproducible were displayed. *P<0.05 versus unstimulated control (–Wnt3a). (C) In vitro methylation assay for G3BP1. F9 cells were transiently transfected with empty vector or HA–PRMT1 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-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 as substrate and [3H]SAM as a methyl donor. Top panel displays the d.p.m. of the incorporated tritiated methyl groups measured from the excised bands of the SDS-PAGE gel and the bottom panel displays a representative fluorograph. (D) In vitro methylation assay for G3BP1 mutants. F9 cells were transiently transfected with HA–PRMT1 for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for 6 h followed by cell lysis and affinity pull-downs with anti-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 and its mutants R433K or R445K as substrates and [3H]SAM as a methyl donor. Representative data of two independent experiments that proved highly reproducible were displayed. (E) In vivo methylation of G3BP1. F9 cells were transiently transfected with empty vector, Myc–G3BP1, or G3BP1 mutants for 24 hours. The cells were then metabolically labeled with L-[methyl-3H]methionine in the presence of protein translation inhibitors. After 3 hours, the cells were lysed and affinity pull-downs with anti-Myc antibodies were performed. The methylation statuses of G3BP1 and its mutants were then revealed by SDS-PAGE followed by fluorography. After fluorography, the gels were rehydrated, transferred to nitrocellulose membranes followed by immunoblotting with anti-Myc antibodies (bottom panel). Representative data of two independent experiments that proved highly reproducible were displayed.
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Figure 3: PRMT1 binds to and methylates G3BP1. (A) Ectopic PRMT1 interacts with G3BP1. F9 cells were transiently transfected with Myc–G3BP1 either alone or with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with anti-HA antibodies. Interaction of G3BP1 with PRMT1 was visualized by probing the blots with anti-Myc antibodies. F9 cells were transiently trasfected with HA–PRMT1 for 24 hours followed by cell lysis and affinity pull-downs with control IgG, anti-Dvl3, anti-GSK3β or anti-HA antibodies. The association of PRMT1 with different proteins was visualized by probing the blots with anti-HA antibodies. (B) F9 cells were transiently transfected with HA–PRMT1 and Myc–G3BP1 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-HA specific 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. Asterisks indicate the bands of Immunoglobulin heavy chain. Representative blots of three independent experiments that proved highly reproducible were displayed. *P<0.05 versus unstimulated control (–Wnt3a). (C) In vitro methylation assay for G3BP1. F9 cells were transiently transfected with empty vector or HA–PRMT1 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-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 as substrate and [3H]SAM as a methyl donor. Top panel displays the d.p.m. of the incorporated tritiated methyl groups measured from the excised bands of the SDS-PAGE gel and the bottom panel displays a representative fluorograph. (D) In vitro methylation assay for G3BP1 mutants. F9 cells were transiently transfected with HA–PRMT1 for 24 hours. The cells were then treated with Wnt3a (20 ng/ml) for 6 h followed by cell lysis and affinity pull-downs with anti-HA specific antibodies followed by an in vitro methylation assay using recombinant GST–G3BP1 and its mutants R433K or R445K as substrates and [3H]SAM as a methyl donor. Representative data of two independent experiments that proved highly reproducible were displayed. (E) In vivo methylation of G3BP1. F9 cells were transiently transfected with empty vector, Myc–G3BP1, or G3BP1 mutants for 24 hours. The cells were then metabolically labeled with L-[methyl-3H]methionine in the presence of protein translation inhibitors. After 3 hours, the cells were lysed and affinity pull-downs with anti-Myc antibodies were performed. The methylation statuses of G3BP1 and its mutants were then revealed by SDS-PAGE followed by fluorography. After fluorography, the gels were rehydrated, transferred to nitrocellulose membranes followed by immunoblotting with anti-Myc antibodies (bottom panel). Representative data of two independent experiments that proved highly reproducible were displayed.
Mentions: Arginine methylation of proteins can be catalyzed by protein arginine methyl transferase 1 (PRMT1), a ubiquitously expressed methyl transferase for histones and other nuclear proteins that bind nucleic acids (Bedford and Clarke, 2009; Lee and Stallcup, 2009). Because G3BP1 was prominently methylated by Wnt3a in the Dvl3-based complex, we evaluated whether PRMT1 was catalyzing the process. Pull-downs performed on F9 cell extracts expressing HA-tagged PRMT1 and Myc-tagged G3BP1 revealed PRMT1–G3BP1 association in these complexes (Fig. 3A). Pull-downsFig. 1.

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