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

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)95-104
Number of pages10
JournalCell structure and function
Volume40
Issue number2
DOIs
Publication statusPublished - 27-06-2015
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Physiology
  • Molecular Biology
  • Cell Biology

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