Bikkavilli RK et al. (2012), Dishevelled3 is a novel arginine methyl transfe...

XB-ART-47072

Sci Rep 2012 Jan 01;2:805. doi: 10.1038/srep00805.

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Dishevelled3 is a novel arginine methyl transferase substrate.

Bikkavilli RK , Avasarala S , Vanscoyk M , Sechler M , Kelley N , Malbon CC , Winn RA .

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Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

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Species referenced: Xenopus
Genes referenced: dvl2 dvl3 myc prmt1 prmt7 wnt3a

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	Figure 1. Dvl3 is a PRMT substrate.(A) Primary amino acid sequences of Dvl3 were scanned for the presence of ‘RG' rich regions and are represented in the figure. (B) Primary amino acid sequences of Dvl3 from mouse, human, Xenopus, zebrafish and Drosophila were aligned using ClustalW. Marked arginine residues are evolutionarily conserved.
	Figure 2. PRMTs catalyze Dvl3 methylation.(A) PRMTs purified from F9 cells were used in an in vitro methylation reaction along with purified myc-hDvl3 (5 µl) and 1 µCi [3H]-SAMe as described in the methods. PRMTs purified from F9 cells treated with Wnt3a (20 ng/ml) were used in an in vitro methylation assay along with Dvl3 purified either from untreated (B) or HEK293 cells treated with Adenosine dialdehyde (C) as described in the methods. (D) F9 cells were labeled with [3H]-L-methyl methionine in the absence or presence of Wnt3a (20 ng/ml) as described in the methods. The methylation status of Dvl3 was revealed by anti-Dvl3 pull-downs, SDS-PAGE and fluorography. After fluorography, the blots were probed with anti-Dvl3 antibodies (lower panel). The amounts of Dvl3 methylated (fluorograph) and the total amount of Dvl3 immunoprecipitated (myc immunoblots) were quantified using Quantity one (BioRad) software and the normalized values (methylated Dvl3/total Dvl3) were represented in the figure as densitometry readings.
	Figure 3. Differential methylation of Dvl3 by PRMT1 and PRMT7.PRMT1 and PRMT7 were purified from F9 cells and were utilized in an in vitro methylation assay using purified myc-hDvl3 (wild-type) or its mutants R271K, R342K, or R614K as substrates and3H-SAMe as a methyl donor.
	Figure 4. R271K mutant of Dvl3 localizes to plasma membrane.(A) HEK293 cells transfected with empty vector, myc-hDvl3 (wild-type) or its mutants R271K, R342K, R614K were fixed, permeabilized and immunostained with anti-myc antibodies and Cy3-labeled secondary antibodies. Scale bar 5 µm. HEK293 cells (B) or bronchial epithelial cells (Beas-2B, C) were transfected either with empty vector, myc-hDvl3 (wild-type) or its methylation-deficient mutants, membrane and cytosolic fractions separated and immunoblots performed with anti-myc and anti-sodium/potassium ATPase antibodies as described in the methods. For quantifying the amounts of wild-type or methylation-deficient mutants of Dvl3 in the membrane fractions of Beas2B cell lysates, Quantity one (BioRad) software was used. Dvl3 amounts were normalized to their corresponding sodium potassium ATPase controls and are represented in the figure as densitometry readings.
	Figure 5. Methylation-deficient mutants of Dvl3 stimulate Lef/Tcf-sensitive gene transcription.HEK293 cells (A) or bronchial epithelial cells (B) were transiently co-transfected with super8xTOPFLASH and either myc-hDvl3 (wild-type) or its methylation-deficient mutants. The lysates were later assayed for luciferase activities as described in the methods. The data represents mean values ± S.E.M. from three independent experiments. **, p< 0.01; versus control (wild-type).

References [+] :

Angers, Proximal events in Wnt signal transduction. 2009, Pubmed