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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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G3BP1 negatively regulates Wnt/β-catenin signaling. (A) F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific to mouse G3bp1 (100 nM) for 48 hours and the levels of mRNA encoding β-catenin and cyclophilin A were quantified using quantitative PCR. The data represent normalized Ctnnb1 mRNA to the Ppia (cyclophilin A) mRNA levels (mean values ± s.e.m.) from two independent experiments whose results were in high agreement. **P<0.01 versus control (control siRNA). F9 cells were treated with either control siRNA (100 nM) or siRNAs specific for mouse G3bp1 (100 nM) for 48 hours and the lysates were assayed either for cytosolic β-catenin levels (B) or Lef/Tcf-sensitive transcription (C). Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a). (D) F9 cells were transfected with Myc–G3BP1 for 24 hours and the lysates were assayed for cytosolic β-catenin stabilization. Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a).
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Figure 4: G3BP1 negatively regulates Wnt/β-catenin signaling. (A) F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific to mouse G3bp1 (100 nM) for 48 hours and the levels of mRNA encoding β-catenin and cyclophilin A were quantified using quantitative PCR. The data represent normalized Ctnnb1 mRNA to the Ppia (cyclophilin A) mRNA levels (mean values ± s.e.m.) from two independent experiments whose results were in high agreement. **P<0.01 versus control (control siRNA). F9 cells were treated with either control siRNA (100 nM) or siRNAs specific for mouse G3bp1 (100 nM) for 48 hours and the lysates were assayed either for cytosolic β-catenin levels (B) or Lef/Tcf-sensitive transcription (C). Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a). (D) F9 cells were transfected with Myc–G3BP1 for 24 hours and the lysates were assayed for cytosolic β-catenin stabilization. Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a).

Mentions: To test whether G3BP1 expression modulated canonical Wnt/β-catenin signaling, Wnt-stimulated β-catenin accumulation and Lef/Tcf-sensitive gene transcription was probed. Knockdown of G3BP1 was effective, in response to treatment with small interfering RNAs (siRNAs). G3BP1 deficiency provoked a twofold increase in basal Ctnnb1 mRNA levels (Fig. 4A). Cells treated with scrambled siRNAs as a control, displayed no such increase (Fig. 4A). G3BP1 deficiency likewise provoked a twofold increase in β-catenin protein levels (Fig. 4B). More telling, G3BP1 deficiency was found to potentiate the ability of Wnt3a to stimulate β-catenin accumulation, the hallmark of canonical Wnt/β-catenin signaling (Fig. 4B). Knockdown of G3BP1 provoked not only an increase in β-catenin protein, but also a consequential increase in Wnt-stimulated Lef/Tcf-sensitive transcription (Fig. 4C). Overexpression of G3BP1 might be expected to attenuate canonical signaling. Indeed, increased expression of G3BP1 attenuated Wnt3a-stimulated β-catenin levels (Fig. 4D).


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

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

G3BP1 negatively regulates Wnt/β-catenin signaling. (A) F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific to mouse G3bp1 (100 nM) for 48 hours and the levels of mRNA encoding β-catenin and cyclophilin A were quantified using quantitative PCR. The data represent normalized Ctnnb1 mRNA to the Ppia (cyclophilin A) mRNA levels (mean values ± s.e.m.) from two independent experiments whose results were in high agreement. **P<0.01 versus control (control siRNA). F9 cells were treated with either control siRNA (100 nM) or siRNAs specific for mouse G3bp1 (100 nM) for 48 hours and the lysates were assayed either for cytosolic β-catenin levels (B) or Lef/Tcf-sensitive transcription (C). Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a). (D) F9 cells were transfected with Myc–G3BP1 for 24 hours and the lysates were assayed for cytosolic β-catenin stabilization. Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a).
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Figure 4: G3BP1 negatively regulates Wnt/β-catenin signaling. (A) F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific to mouse G3bp1 (100 nM) for 48 hours and the levels of mRNA encoding β-catenin and cyclophilin A were quantified using quantitative PCR. The data represent normalized Ctnnb1 mRNA to the Ppia (cyclophilin A) mRNA levels (mean values ± s.e.m.) from two independent experiments whose results were in high agreement. **P<0.01 versus control (control siRNA). F9 cells were treated with either control siRNA (100 nM) or siRNAs specific for mouse G3bp1 (100 nM) for 48 hours and the lysates were assayed either for cytosolic β-catenin levels (B) or Lef/Tcf-sensitive transcription (C). Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. *P<0.05; **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a). (D) F9 cells were transfected with Myc–G3BP1 for 24 hours and the lysates were assayed for cytosolic β-catenin stabilization. Top panel displays mean values ± s.e.m. obtained from three independent experiments; the bottom panel displays representative blots. **P<0.01 versus unstimulated control (–Wnt3a). ##P<0.01 versus stimulated control (+Wnt3a).
Mentions: To test whether G3BP1 expression modulated canonical Wnt/β-catenin signaling, Wnt-stimulated β-catenin accumulation and Lef/Tcf-sensitive gene transcription was probed. Knockdown of G3BP1 was effective, in response to treatment with small interfering RNAs (siRNAs). G3BP1 deficiency provoked a twofold increase in basal Ctnnb1 mRNA levels (Fig. 4A). Cells treated with scrambled siRNAs as a control, displayed no such increase (Fig. 4A). G3BP1 deficiency likewise provoked a twofold increase in β-catenin protein levels (Fig. 4B). More telling, G3BP1 deficiency was found to potentiate the ability of Wnt3a to stimulate β-catenin accumulation, the hallmark of canonical Wnt/β-catenin signaling (Fig. 4B). Knockdown of G3BP1 provoked not only an increase in β-catenin protein, but also a consequential increase in Wnt-stimulated Lef/Tcf-sensitive transcription (Fig. 4C). Overexpression of G3BP1 might be expected to attenuate canonical signaling. Indeed, increased expression of G3BP1 attenuated Wnt3a-stimulated β-catenin levels (Fig. 4D).

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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