Identification of <i>C</i>-mannosylation in a receptor tyrosine kinase AXL

C-mannosylation is a unique type of glycosylation in which a mannose is added to tryptophan in a protein. However, the biological function of C-mannosylation is still largely unknown. AXL is a receptor tyrosine kinase, and its overexpression contributes to tumor malignancy. The role of AXL in cancer cells is broad, including invasion, drug resistance, and vasculogenic mimicry formation. Although Trp320 of AXL was predicted to be C-mannosylated, it has not been confirmed. Here, we demonstrated that Trp320 of AXL is C-mannosylated, measured by mass spectrometry of recombinant AXL purified from various cancer cells. Furthermore, re-expression of C-mannosylation-deficient AXL in human breast cancer MDA-MB-231 cells lacking AXL by the CRISPR/Cas9 system resulted in reduction of vasculogenic mimicry formation. Interestingly, phosphorylation levels of AKT in C-mannosylation-deficient AXL re-expressing cells were comparable to those of parental and wild-type AXL re-expressing cells. These results represent the first discovery of C-mannosylation in a receptor tyrosine kinase and the possibility that C-mannosylation may affect AXL function, distinct from its downstream signaling in cancer cells.