Quantitative Tyrosine Phosphoproteome Profiling of AXL Receptor Tyrosine Kinase Signaling Network

SIMPLE SUMMARY: AXL is a receptor tyrosine kinase belonging to the TAM (Tyro3, Axl and Mer) family. The AXL protein plays an important role in promoting cancer development, such as proliferation, migration, invasion and survival of cancer cells. In this study, we used mass spectrometry-based proteomics to quantify the cancer signaling regulated by AXL activation. Our study identified more than 1000 phosphotyrosine sites and discovered that activation of AXL can upregulate multiple cancer-promoting and cell migration/invasion-related signaling pathways. We also observed significant crosstalk as evidenced by rapid phosphorylation of multiple receptor tyrosine kinases and protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. These discoveries should serve as a potentially useful resource for studying AXL functions as well as for the development of effective therapeutic options to target AXL. ABSTRACT: Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.