Looking for novel interactors of IL-13 receptor α2 (IL13Rα2) and tyrosine-protein phosphatase non-receptor type 1 (PTPN1/ PTP1B) by using proximity-dependent biotinylation (BioID)

Abstract

Introduction: IL13Rα2 is a high affinity receptor for IL-13 that is re-expressed in late neoplastic stages. The IL- 13/IL- 13Rα2 axis triggers invasion in colorectal cancer (CRC) and other cancers in a process mediated, among others, by the phosphatase PTPN1 (aka PTP1B), involved in the dephosphorylation of the autoinhibitory Src Tyr416, and further Src activation. PTPN1 takes part in IL13Rα2 internalization and associates with poor outcome in glioblastoma and CRC patients. Inhibition of IL13Rα2 pathway decreases invasion of multiple cancer cell lines (i.e., SKOV-3, PC-3 and KM12SM). To better define the oncogenic properties of IL13Rα2 and PTPN1, we aim to identify novel interactors by using proximity-dependent biotinylation coupled to mass spectrometry. Materials and methods: PTPN1 and IL13Rα2 were cloned into pcDNA.5-pDEST-BirA-Flag-Ct by using LR clonase enzyme, obtaining the desired plasmids containing fusion genes PTPN1-BirA and IL13Rα2-BirA. Flp-In™ T-REx™ cells were co-transfected with pOG44 and pcDNA.5 vectors and selected with hygromycin. Then doxycycline and biotin were added to induce the expression of the fusion gene and proximity labelling. Biotinylated proteins were purified by affinity using streptavidin-sepharose, trypsin-digested and analysed in a Q-Exactive mass spectrometer. Raw data were analysed with MaxQuant, followed by Perseus for statistical analysis and SAINT for probabilistic scoring of protein-protein interaction data, which were subjected to gene ontology (GO) analysis. Results: For IL13Rα2, 735 proteins were identified and 94 were scored as significant interactors by SAINT. GO enrichment analysis showed association with cadherin binding and response to reticulum stress, among others. 1009 proteins were identified and 217 were scored as significant PTPN1 interactors. GO enrichment analysis showed important association with mitotic cell cycle regulation, such as CDK1 (G2/M transition) or PCTN (centrosome component). Among the 19 IL13Rα2 and PTPN1 common interactors stand out those related to SNARE complex (YKT6, SNAP29 and VAMP3), which is involved in regulation of cancer invasion, drug resistance and kinase phosphorylation. Another interesting finding was the presence of VRK2, a kinase involved in MAPK pathway. Conclusions: This study reveals novel individual interactors of IL13Rα2 and PTPN1, but also identifies common interactors of both proteins such as members of the SNARE complex and VRK2, opening new strategies for the inhibition of IL13Rα2 and PTPN1 in cancer metastasis.