Abstract
Asparagine-linked glycosylation is an endoplasmic reticulum co- and post- translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.
Highlights
Receptor tyrosine kinases (RTKs) are key regulators of several critical biological processes including cell proliferation, migration, cell survival, and cell-cycle control [1,2,3,4,5]
While the results show that mannose phosphate isomerase (MPI) function is required for activation of FGFR2 and ErbB3, they indicate that EGFR phosphorylation was not affected by MPI knockdown, and suggest that the functionality of FGFR is sensitive to low levels of MPI activity
The successful establishment of a MPI deficient cell line provides a novel tool for glycobiology studies as few mammalian cell lines with N-linked glycosylation (NLG) gene defects exist and MPI deficient models are otherwise limited to fibroblasts derived from patients with congenital disorders of glycosylation type IB (CDG1B) [19,20,29,30,31,32,33] These immortalized fibroblasts grow relatively slowly in culture but have no explicit phenotype with respect to the expression or function of specific proteins [19,31
Summary
Receptor tyrosine kinases (RTKs) are key regulators of several critical biological processes including cell proliferation, migration, cell survival, and cell-cycle control [1,2,3,4,5]. RTKs are typically activated via ligand-induced dimerization, which stimulates kinase activation, and trans-phosphorylation of the carboxy-terminal tail, creating docking sites for phosphotyrosine-binding effectors that initiate intracellular signaling cascades. Because of their broad roles in many crucial cellular processes, RTK activation is normally under tight control, and loss of regulatory mechanisms lead to the genesis and progression of a variety of human cancers [2,3,7]. FGFRs are a major focus for the development of new therapeutic approaches in patients whose tumors harbor enhanced FGF/ FGFR signaling [4,7,9], and understanding the unique mechanisms that regulate this receptor family will be important for translating these targeted approaches to the clinic
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
