Annual Report 2024

Laboratory of Intracellular Traffic and Oncology

Yuuki Obata, Sho Ichikawa, Takuma Okamoto, Risako Yunokihara, Naoe Takayama

Introduction

 Gain-of-function mutations in receptor protein-tyrosine kinases (RTKs) are critical drivers for development of cancer. Considering that in normal cells, wild-type RTKs are localized to the plasma membrane (PM) to bind their ligands, RTK mutants have been thought to be distributed on the cell surface membrane. However, we found that mutant KIT RTK is aberrantly localized in the intracellular compartments such as the Golgi/trans-Golgi network (TGN), endoplasmic reticulum (ER) and endosomes. It predominantly causes downstream activations in the endomembranes. Our investigations have also identified the presence of other mutant proteins in organelles. Therefore, mislocalized growth signals in organelles are a characteristic feature of cancer-causing RTK mutants. Our primary aim is to clarify the molecular mechanism underlying RTK retention in organelles and to develop a new strategy for the suppression of oncogenic RTK signaling.

The Team and What We Do

 We mainly perform spatio-temporal analyses of tyrosine phosphorylation signaling in cancer cells with fluorescent imaging technics and biochemical assays, such as immunoblotting, pulldown, and immunoprecipitation.

Research Activities

Molecular mechanism of ER export of RTKs

 Inhibitors of protein export from the ER, such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, indicating that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs, small GTPase) that play a crucial role in ER–Golgi protein trafficking. M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. Single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, suggesting that BFA/M-COPA treatment cannot be mimicked by knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Thus, these results suggest that RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export (Natsume et al., 2024, JBC). Trafficking inhibition from the ER would be a promising strategy for the suppression of growth signaling of RTK.

Mechanism of the retention of KIT RTK in the Golgi/TGN in gastrointestinal stromal tumor (GIST)

 Recently, we discovered that, during biosynthetic transport, the KIT mutant (KIT^mut) is retained in the Golgi/TGN, where it activates downstream molecules. This retention is dependent on the phospholipase Cγ2–protein kinase D2–PI4 kinase IIIβ (PLCγ2–PKD2–PI4KIIIβ) pathway, which KIT^mut activates at the Golgi/TGN. The activated cascade aberrantly recruits GGA1 and the γ-adaptin subunit of AP1, resulting in KIT^mut retention in the Golgi/TGN. However, the precise mechanisms, including the mediators and effectors of the pathway, remain unclear. In humans, the phosphatidic acid-generating enzymes, phospholipase D1 (PLD1) and PLD2, are known downstream proteins of PKD. In the presence of a PLD inhibitor, KIT^mut is released from the Golgi/TGN and subsequently degraded in lysosomes, leading to signal inactivation. Knockdown experiments indicated that PLD2 plays a role in KIT^mut retention. KIT^mut activates PLD2 through PKD2, but not PI4KIIIβ, for Golgi/TGN retention. PLD activity is required for the association of γ-adaptin with GGA1. Therefore, the KIT–PLCγ2–PKD2 pathway separately activates PLD2 and PI4KIIIβ to recruit γ-adaptin and GGA1. Collectively, these results suggest that KIT^mut retention is dependent on the activation of the PLCγ2–PKD2–PLD2 cascade in GIST cells (Obata et al., 2025, bioRxiv).

Education

 Teaching students (one graduate student, two undergraduate students)

Future Prospects

 Because loss-of-function of PLD2 does not affect the localization and signaling of RTKs other than KIT, such as FLT3-ITD, further studies are required to understand the mechanism of Golgi retention of individual oncogenic signaling molecules. Furthermore, we found that blockade of ER export of RTK mutants markedly suppresses growth signaling. These studies suggest that understanding the mechanism of intracellular trafficking will help us establish a novel method for the suppression of cancer cell proliferation.

List of papers published in 2024

Journal

1. Obata Y, Natsume M, Shiina I, Takahashi T, Nishida T. Golgi retention of KIT in gastrointestinal stromal tumour cells is phospholipase D activity-dependent. bioRxiv, 2025

2. Natsume M, Niwa M, Ichikawa S, Okamoto T, Tsutsui H, Usukura D, Murata T, Abe R, Shimonaka M, Nishida T, Shiina I, Obata Y. Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5. The Journal of biological chemistry, 300:107327, 2024