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タイトル: | Validity of the cell‐extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases |
著者: | Niinae, Tomoya Sugiyama, Naoyuki Ishihama, Yasushi https://orcid.org/0000-0001-7714-203X (unconfirmed) |
著者名の別形: | 新苗, 智也 杉山, 直幸 石濱, 泰 |
キーワード: | Kinase substrate Phosphoproteomics Phosphorylation motif Protein kinase |
発行日: | Oct-2023 |
出版者: | Molecular Biology Society of Japan Wiley |
誌名: | Genes to Cells |
巻: | 28 |
号: | 10 |
開始ページ: | 727 |
終了ページ: | 735 |
抄録: | Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest. |
著作権等: | © 2023 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
URI: | http://hdl.handle.net/2433/287218 |
DOI(出版社版): | 10.1111/gtc.13063 |
PubMed ID: | 37658684 |
出現コレクション: | 学術雑誌掲載論文等 |
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