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

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.