Phosphoproteomic analysis using the WW and FHA domains as biological filters

Md Hasanuzzaman Shohag, Tomoki Nishioka, Rijwan Uddin Ahammad, Shinichi Nakamuta, Yoshimitsu Yura, Tomonari Hamaguchi, Kozo Kaibuchi, Mutsuki Amano

研究成果: Article査読

7 被引用数 (Scopus)

抄録

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 Pinl-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.

本文言語English
ページ(範囲)95-104
ページ数10
ジャーナルCell structure and function
40
2
DOI
出版ステータスPublished - 27-06-2015
外部発表はい

All Science Journal Classification (ASJC) codes

  • 生理学
  • 分子生物学
  • 細胞生物学

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