title: Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters.
abstract: Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.
authors.affiliation: Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine.
authors.forename: Md Hasanuzzaman
authors.initials: MH
authors.lastname: Shohag
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Rijwan Uddin
authors.initials: RU
authors.lastname: Ahammad
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nakamuta
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Yura
authors.forename: Tomonari
authors.initials: T
authors.lastname: Hamaguchi
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry*; D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008387:Marine Toxins; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
chemical_list: D048948:14-3-3 Proteins; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; C572235:YWHAZ protein, human; D005576:Colforsin; D019319:Okadaic Acid; C059041:calyculin A