Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters
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- Hasanuzzaman Shohag Md.
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Nishioka Tomoki
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Uddin Ahammad Rijwan
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Nakamuta Shinichi
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Yura Yoshimitsu
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Hamaguchi Tomonari
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Kaibuchi Kozo
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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- Amano Mutsuki
- Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine
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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 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.
収録刊行物
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- Cell Structure and Function
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Cell Structure and Function 40 (2), 95-104, 2015
日本細胞生物学会
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詳細情報 詳細情報について
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- CRID
- 1390282679671568384
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- NII論文ID
- 130005094036
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- NII書誌ID
- AA0060007X
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- ISSN
- 13473700
- 03867196
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- NDL書誌ID
- 027281722
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- PubMed
- 26119529
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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- 使用不可