Imatinib-Induced Changes in Protein Expression and ATP-Binding Affinities of Kinases in Chronic Myelocytic Leukemia Cells

Abstract

Kinases are crucial components in numerous cell signaling pathways. Aberrant expression and activation of protein kinases are known to be accompanied by many types of cancer, and more than 30 small-molecule kinase inhibitors have been approved by the Food and Drug Administration (FDA) for cancer chemotherapy. Biological and clinical applications of small-molecule kinase inhibitors require comprehensive characterizations about how these inhibitors modulate the protein expression and activities of kinases at the entire proteome scale. In this study, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method to monitor the alterations in protein expression of kinases in K-562 chronic myelocytic leukemia (CML) cells elicited by treatment with imatinib, an ABL kinase inhibitor approved by the FDA for CML treatment. By employing isotope-coded ATP affinity probes together with liquid chromatography-multiple-reaction monitoring (LC-MRM) analysis, we also examined the modulation of the ATP-binding affinities of kinases induced by imatinib treatment. The results revealed profound increases in protein expression levels of a large number of kinases in K-562 cells upon treatment with imatinib, which is accompanied by substantial decreases in ATP-binding capacities of many kinases. Apart from ABL kinases, we identified a number of other kinases whose ATP-binding affinities are markedly diminished upon imatinib treatment, including CHK1, a checkpoint kinase involved in DNA damage response signaling. Together, our targeted quantitative proteomic methods enabled, for the first time, dual assessments of small-molecule kinase inhibitor-induced changes in protein expression and ATP-binding affinities of kinases in live cells.

A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis

“…Our primary objective was to explore the potential roles of kinases in melanoma metastasis. Toward this end, we applied a PRM-based targeted proteomic method 12 , in combination with SILAC 19 , to examine the differences in kinase protein expression in 3 pairs of primary/metastatic melanoma cells (i.e. IGR-39/IGR-37, WM-115/WM-266-4, and WM-793/1205Lu) ( Fig.…”

“…For example, isotope-coded ATP-affinity probes were shown to be effective in the covalent labeling and enrichment of kinase proteins or their component peptides for subsequent LC-MS/MS analysis [9][10][11] . Both the protein expression level of a kinase and its ATP-binding affinity can affect the extent of kinase labeling by the ATP affinity probe, where the ATP binding affinity of a kinase is sometimes modulated by its activity 12 . Additionally, affinity resin immobilized with various kinase inhibitors was utilized for kinase protein enrichment, where the enrichment efficiency may also be impacted by kinase activity [13][14][15] .…”

“…Additionally, affinity resin immobilized with various kinase inhibitors was utilized for kinase protein enrichment, where the enrichment efficiency may also be impacted by kinase activity [13][14][15] . Recently, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method for assessing specifically the kinase protein expression at the entire proteome scale 12 . In this regard, we established a Skyline LC-PRM library for kinome analysis based on the shotgun proteomic data collected by analyzing tryptic digestion mixtures from multiple human cell lines 12 .…”

“…In this regard, we established a Skyline LC-PRM library for kinome analysis based on the shotgun proteomic data collected by analyzing tryptic digestion mixtures from multiple human cell lines 12 . The library encompasses 1050 tryptic peptides representing 478 kinase proteins, and 395 of them are protein kinases 12,[16][17][18] .…”

“…In this work, we applied the recently established PRM-based targeted proteomic method 12 , in combination with stable isotope labeling by amino acids in cell culture (SILAC), to examine the differential protein expression of kinases in 3 pairs of matched primary/metastatic melanoma cells. We were able to quantify the relative expression levels of approximately 300 unique kinase proteins in each pair of cell lines.…”