Validity of the cell-extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases.
Kinase substrate
Phosphoproteomics
Phosphorylation motif
Protein kinase
Journal
Genes to cells : devoted to molecular & cellular mechanisms
ISSN: 1365-2443
Titre abrégé: Genes Cells
Pays: England
ID NLM: 9607379
Informations de publication
Date de publication:
Oct 2023
Oct 2023
Historique:
revised:
08
08
2023
received:
01
07
2023
accepted:
21
08
2023
medline:
9
10
2023
pubmed:
2
9
2023
entrez:
2
9
2023
Statut:
ppublish
Résumé
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.
Substances chimiques
Proteome
0
Cell Extracts
0
Protein Kinases
EC 2.7.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
727-735Subventions
Organisme : Japan Agency for Medical Research and Development
ID : JP18gm1010010
Organisme : Japan Science and Technology Corporation
ID : JPMJCR1862
Organisme : Japan Society for the Promotion of Science
ID : 17H03605
Organisme : Japan Society for the Promotion of Science
ID : 18H04799
Organisme : Japan Society for the Promotion of Science
ID : 20H04845
Organisme : Japan Society for the Promotion of Science
ID : 21H02459
Organisme : Japan Society for the Promotion of Science
ID : 21H02466
Organisme : Japan Society for the Promotion of Science
ID : 21J15068
Organisme : Japan Society for the Promotion of Science
ID : 23H04924
Informations de copyright
© 2023 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
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