Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5

Miyuki Natsume; Mariko Niwa; Sho Ichikawa; Takuma Okamoto; Hisazumi Tsutsui; Daiki Usukura; Takatsugu Murata; Ryo Abe; Motoyuki Shimonaka; Toshirou Nishida; Isamu Shiina; Yuuki Obata

doi:10.1016/j.jbc.2024.107327

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5

J Biol Chem. 2024 Apr 26;300(6):107327. doi: 10.1016/j.jbc.2024.107327. Online ahead of print.

Authors

Affiliations

¹ Laboratory of Intracellular Traffic & Oncology, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; Faculty of Science, Department of Applied Chemistry, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan.
² Laboratory of Intracellular Traffic & Oncology, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; Faculty of Science, Department of Chemistry, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan.
³ Faculty of Science, Department of Applied Chemistry, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan.
⁴ Tokyo University of Science, Noda, Chiba, Japan.
⁵ Faculty of Science, Department of Chemistry, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan.
⁶ National Cancer Center Hospital, Chuo-ku, Tokyo, Japan; Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan.
⁷ Laboratory of Intracellular Traffic & Oncology, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan. Electronic address: yuobata@ncc.go.jp.

PMID: 38679330
DOI: 10.1016/j.jbc.2024.107327

Abstract

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

Keywords: ARF; BFA; EGFR; GEF; Golgi/TGN; KIT; M-COPA; MET; RTK; endoplasmic reticulum.