O-mannosylation and furin processing are critical maturation events for the KIAA1549 protein implicated in pediatric pilocytic astrocytomas

Abstract

The gene fusion KIAA1549::BRAF is a common oncogenic driver in pediatric pilocytic astrocytoma. The chimeric fusion protein that comprises most of the type 1 transmembrane KIAA1549 protein fused in its C-terminal cytosolic domain to the truncated BRAF oncogene without its N-terminal autoinhibitory domains. KIAA1549 has a large N-terminal ectodomain that undergoes extensive O-mannosylation directed by the POMT1/2 protein O-mannosyltransferase complex localized in the ER. Here, we investigated how posttranslational modifications contribute to KIAA1549 protein maturation in the secretory pathway. Using a panel of KIAA1549 expression constructs and glycoengineered HEK293 cells, we provide evidence that O-mannosylation of the KIAA1549 ectodomain is required for ER exit, acquisition of complex N-glycans, and subsequent furin-like cleavage in the Golgi. In the absence of POMT1/2 O-mannosylation KIAA1549 is retained in ER and fails to undergo proprotein processing. Our findings confirm and extend our previous studies with the KIAA1549::BRAF fusion protein, where oncogenic transformation was dependent on POMT1/2 O-mannosylation and affected on furin processing. Thus, these intrinsic posttranslational modifications of the KIAA1549 ectodomain are critical determinants of the cellular trafficking that lead to activation of the BRAF oncogene, potentially involving a cascade of cleavage events in the Golgi that ultimately release a truncated but fully active BRAF kinase domain into the cytosol.