N-Glycosylation of AXL Receptor Tyrosine Kinase Regulates Its Stability, Phosphorylation, and Oncogenic Function

AXL, a receptor tyrosine kinase implicated in tumor progression, undergoes post-translational modifications that regulate its activity and stability. In this study, we demonstrated that AXL is extensively N-glycosylated in breast and ovarian cancer cells, existing predominantly as two isoforms corresponding to high-mannose and complex-type glycans. The extracellular cleaved soluble form of AXL (sAXL) primarily carries complex N-glycans and relies on glycosylation maturation. Functional analyses showed that complex glycosylation is essential for AXL membrane translocation and phosphorylation, as inhibition of N-glycan maturation caused cytoplasmic accumulation and impaired receptor activation. Mass spectrometry-based glycoproteomic analysis identified five glycosylation sites (N43, N157, N198, N339, and N345) with extensive microheterogeneity, including sialylation, fucosylation, and bisecting GlcNAc modifications. Evolutionary conservation analysis and mutational studies revealed that glycosylation at N43 and N339 is critical for AXL phosphorylation and cell proliferation, while multiple sites collectively contribute to protein stability and subcellular trafficking. Overall, our results demonstrate that N-glycosylation is essential for AXL stability, localization, and oncogenic signaling, offering new insights into the role glycosylation in regulating receptor tyrosine kinases function in cancer.