title: Delicate analysis of post-translational modifications on Dishevelled 3.
abstract: The Wnt/β-catenin signaling pathway plays a critical role in multiple developmental events during embryogenesis and adult tissue homeostasis. Dishevelled (Dvl) is an important component of Wnt/β-catenin signaling pathway, although the modification, especially phosphorylation, seems closely related to the activity and stability of Dvl, the overall modification status of Dvl is still poorly understood. In this study, we focused on low abundant Dvl3, one of the three Dvl isoforms. Using affinity purification of different sources of Dvl3, followed by digestion with both trypsin and chymotrypsin, we systematically analyzed the overall modification status of Dvl3 with liquid chromatography coupled LTQ-Orbitrap. Altogether, we confidently identified Dvl3 with more than 50% sequence coverage, including 6 phosphorylation, 4 methylation, and 1 dimethylation sites. Most of the identified modification sites were novel and located in the linker region between different motifs. Subsequently, the validity of modified peptides was confirmed by synthetic modified peptides. Finally, SRM analysis was performed to verify and quantify the successive change of one dimethylation site on Dvl3 in vivo during Wnt signaling. Taken together, our study elucidated the existence of a novel post-translational modification on Dvl3 and provided a delicate experimental procedure for analysis of modification on low abundant proteins.
authors.affiliation: Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Chaochao
authors.initials: C
authors.lastname: Wu
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.forename: Qingrun
authors.initials: Q
authors.lastname: Li
authors.forename: Quanhu
authors.initials: Q
authors.lastname: Sheng
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000737:chemistry; D000072261:Dishevelled Proteins; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008745:Methylation; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C000606584:DVL3 protein, human; D000072261:Dishevelled Proteins; D010750:Phosphoproteins; D002918:Chymotrypsin