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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 binds Ctnnb1 mRNA. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, full-length G3BP1 (1–465), its N-terminal half (1–240) or its C-terminal half (241–465) with anti-myc antibodies. Ctnnb1 mRNA in the immunoprecipitates was quantified using quantitative PCR. The top panel represents mean values ± s.e.m. obtained from two independent experiments; the bottom panel displays representative gel picture of two independent experiments that proved highly reproducible. **P<0.01 versus control (pCMV-Myc). (B,C) Northwestern analysis of interaction of G3BP1 with Ctnnb1 mRNA. Recombinant GST or GST–G3BP1 (B) or immunoprecipitated Myc–G3BP1 from F9 cell lysates (C) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Northwestern analysis was then performed either using DIG-labeled Ctnnb1 UTR or Gapdh UTR probes. The top panels represent northwestern blots whereas the lower panels display either Ponceau S staining (B) or immunoblotting with anti-Myc antibodies for the same blots. (D–F) Identification of G3BP1 binding region within the 3′-UTR of Ctnnb1 mRNA. Immunoprecipitated Myc–G3BP1 from F9 cell lysates were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed using truncated versions of DIG-labeled Ctnnb1 UTR probes. The top panels represent northwestern blots, whereas lower panels display immunoblots with anti-Myc antibodies. Representative data of two independent experiments that proved highly reproducible are displayed.
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Figure 5: G3BP1 binds Ctnnb1 mRNA. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, full-length G3BP1 (1–465), its N-terminal half (1–240) or its C-terminal half (241–465) with anti-myc antibodies. Ctnnb1 mRNA in the immunoprecipitates was quantified using quantitative PCR. The top panel represents mean values ± s.e.m. obtained from two independent experiments; the bottom panel displays representative gel picture of two independent experiments that proved highly reproducible. **P<0.01 versus control (pCMV-Myc). (B,C) Northwestern analysis of interaction of G3BP1 with Ctnnb1 mRNA. Recombinant GST or GST–G3BP1 (B) or immunoprecipitated Myc–G3BP1 from F9 cell lysates (C) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Northwestern analysis was then performed either using DIG-labeled Ctnnb1 UTR or Gapdh UTR probes. The top panels represent northwestern blots whereas the lower panels display either Ponceau S staining (B) or immunoblotting with anti-Myc antibodies for the same blots. (D–F) Identification of G3BP1 binding region within the 3′-UTR of Ctnnb1 mRNA. Immunoprecipitated Myc–G3BP1 from F9 cell lysates were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed using truncated versions of DIG-labeled Ctnnb1 UTR probes. The top panels represent northwestern blots, whereas lower panels display immunoblots with anti-Myc antibodies. Representative data of two independent experiments that proved highly reproducible are displayed.

Mentions: G3BP1 is known to bind the 3′-untranslated regions (3′-UTRs) of Myc or Tau mRNAs (Gallouzi et al., 1998; Liu et al., 1999; Tourriere et al., 2001; Atlas et al., 2004; Atlas et al., 2007). Ctnnb1 mRNA is present in Dvl3-based complexes (Bikkavilli and Malbon, 2010). Because it has domains necessary for RNA binding, G3BP1 was tested for its ability to bind Ctnnb1 mRNA. The presence of Ctnnb1 mRNAs in the G3BP1 complex was probed. Myc–G3BP1 and its N-terminal or C-terminal complexes were isolated from cell lysates by immunoprecipitation with anti-Myc antibodies. RNA was isolated from the pull-downs and amplified by RT-PCR. Ctnnb1 transcripts were found in the G3BP1 complex (Fig. 5A). Pull-downs prepared from lysates of cells transfected with empty pCMV vector, by contrast, displayed no detectable Ctnnb1 mRNA (Fig. 5A). Pull-downs performed with cells expressing the C-terminal region of G3BP1 (241–465) also displayed Ctnnb1 transcripts (Fig. 5A). However, pull-downs performed with cells expressing the N-terminal region (1–240 amino acids) of G3BP1, did not display any Ctnnb1 mRNA (Fig. 5A). Therefore, G3BP1 appears to bind and regulate Ctnnb1 mRNA.


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

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

G3BP1 binds Ctnnb1 mRNA. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, full-length G3BP1 (1–465), its N-terminal half (1–240) or its C-terminal half (241–465) with anti-myc antibodies. Ctnnb1 mRNA in the immunoprecipitates was quantified using quantitative PCR. The top panel represents mean values ± s.e.m. obtained from two independent experiments; the bottom panel displays representative gel picture of two independent experiments that proved highly reproducible. **P<0.01 versus control (pCMV-Myc). (B,C) Northwestern analysis of interaction of G3BP1 with Ctnnb1 mRNA. Recombinant GST or GST–G3BP1 (B) or immunoprecipitated Myc–G3BP1 from F9 cell lysates (C) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Northwestern analysis was then performed either using DIG-labeled Ctnnb1 UTR or Gapdh UTR probes. The top panels represent northwestern blots whereas the lower panels display either Ponceau S staining (B) or immunoblotting with anti-Myc antibodies for the same blots. (D–F) Identification of G3BP1 binding region within the 3′-UTR of Ctnnb1 mRNA. Immunoprecipitated Myc–G3BP1 from F9 cell lysates were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed using truncated versions of DIG-labeled Ctnnb1 UTR probes. The top panels represent northwestern blots, whereas lower panels display immunoblots with anti-Myc antibodies. Representative data of two independent experiments that proved highly reproducible are displayed.
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Figure 5: G3BP1 binds Ctnnb1 mRNA. (A) RNA immunoprecipitation assay was performed on F9 cell lysates expressing either empty vector, full-length G3BP1 (1–465), its N-terminal half (1–240) or its C-terminal half (241–465) with anti-myc antibodies. Ctnnb1 mRNA in the immunoprecipitates was quantified using quantitative PCR. The top panel represents mean values ± s.e.m. obtained from two independent experiments; the bottom panel displays representative gel picture of two independent experiments that proved highly reproducible. **P<0.01 versus control (pCMV-Myc). (B,C) Northwestern analysis of interaction of G3BP1 with Ctnnb1 mRNA. Recombinant GST or GST–G3BP1 (B) or immunoprecipitated Myc–G3BP1 from F9 cell lysates (C) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Northwestern analysis was then performed either using DIG-labeled Ctnnb1 UTR or Gapdh UTR probes. The top panels represent northwestern blots whereas the lower panels display either Ponceau S staining (B) or immunoblotting with anti-Myc antibodies for the same blots. (D–F) Identification of G3BP1 binding region within the 3′-UTR of Ctnnb1 mRNA. Immunoprecipitated Myc–G3BP1 from F9 cell lysates were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Northwestern analysis was then performed using truncated versions of DIG-labeled Ctnnb1 UTR probes. The top panels represent northwestern blots, whereas lower panels display immunoblots with anti-Myc antibodies. Representative data of two independent experiments that proved highly reproducible are displayed.
Mentions: G3BP1 is known to bind the 3′-untranslated regions (3′-UTRs) of Myc or Tau mRNAs (Gallouzi et al., 1998; Liu et al., 1999; Tourriere et al., 2001; Atlas et al., 2004; Atlas et al., 2007). Ctnnb1 mRNA is present in Dvl3-based complexes (Bikkavilli and Malbon, 2010). Because it has domains necessary for RNA binding, G3BP1 was tested for its ability to bind Ctnnb1 mRNA. The presence of Ctnnb1 mRNAs in the G3BP1 complex was probed. Myc–G3BP1 and its N-terminal or C-terminal complexes were isolated from cell lysates by immunoprecipitation with anti-Myc antibodies. RNA was isolated from the pull-downs and amplified by RT-PCR. Ctnnb1 transcripts were found in the G3BP1 complex (Fig. 5A). Pull-downs prepared from lysates of cells transfected with empty pCMV vector, by contrast, displayed no detectable Ctnnb1 mRNA (Fig. 5A). Pull-downs performed with cells expressing the C-terminal region of G3BP1 (241–465) also displayed Ctnnb1 transcripts (Fig. 5A). However, pull-downs performed with cells expressing the N-terminal region (1–240 amino acids) of G3BP1, did not display any Ctnnb1 mRNA (Fig. 5A). Therefore, G3BP1 appears to bind and regulate Ctnnb1 mRNA.

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