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Validity of the cell-extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases

ABSTRACT: Three representative protein kinases with different substrate preference including ERK1 (pro-directed), CK2 (acidophilic), and PKA (basophilic) and the substrate pool proteome from three different cell lines including MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), Jurkat (human acute T-cell leukemia) were used to evaluate phosphorylation sequence motifs. Specifically, recombinant kinases were added to the cell-extracted proteome, phosphorylated in vitro, trypsin digested, and the phosphopeptides were enriched for 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. The results showed that each kinase exhibited identical phosphorylation motifs, independent of the substrate proteome. Furthermore, the motifs were also consistent with those from completely randomized peptide libraries. 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 the phosphorylation motifs from the phosphoproteome analysis of cell extracts as a substrate pool for the kinase of interest.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD042871 | JPOST Repository | Tue Jun 11 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	180708TN_02_HeLa_F1.raw	Raw
	180708TN_03_HeLa_F2.raw	Raw
	180708TN_04_HeLa_F3.raw	Raw
	180708TN_05_HeLa_F4.raw	Raw
	180708TN_06_HeLa_F5.raw	Raw

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