Tae, Kun Cho, Sang Woo Lee, Seonjeong Heo, Dahyoon Cha, Hyo Sun Lee, Da Yeon Oh, Eunjeong Choi, Minhyeong Shin, Donghyuk Yang, Siyoung Lee, Cheolju Choi, Cheol Yong 2025-11-26T10:01:57Z 2025-11-26T10:01:57Z 2025-11-26 2025-11 2041-4889 https://pubs.kist.re.kr/handle/201004/153669 The TGF-β signaling pathway is initiated when the type II receptor phosphorylates the type I receptor (ALK5) upon TGF-β binding. While E3 ubiquitin ligases regulate TGF-β receptor degradation, their role in modulating receptor catalytic activity via ubiquitination remains largely unexplored. Here, we demonstrate that the E3 ubiquitin ligase MARCH2 enhances ALK5 catalytic activity by conjugating K63-linked ubiquitin chains to lysines 342/343 (K342/343), primarily at endosomes following TGF-β-induced endocytosis. Mutations of ALK5 at K342/343 (K342/343R) abolish its catalytic activity for SMAD2 phosphorylation, leading to impaired TGF-β responses and reduced cell migration in A549 cells. In a mouse model, expression of the ALK5 K342/343 R mutant significantly decreases lung metastasis compared to wild-type ALK5. TCGA analysis further revealed a strong positive correlation between MARCH2 expression and TGF-β target gene expression. Collectively, these findings establish ALK5 ubiquitination at K342/343 by MARCH2 as a crucial regulatory mechanism for ALK5 catalytic activity, TGF-β signaling, and metastasis. English Nature Publishing Group MARCH2-mediated Lys63-linked polyubiquitination promotes metastasis by modulating the catalytic activity of TGF-β type I receptor Article 10.1038/s41419-025-08145-3 1 Cell Death & Disease, v.16, no.1 Cell Death & Disease 16 1 Y scie scopus 001611548700012 2-s2.0-105021290085 Cell Biology Cell Biology Article MARCH LIGASES FYVE DOMAIN PROTEIN UBIQUITINATION ENDOCYTOSIS ACTIVATION ANCHOR SMAD2 SARA EARLY ENDOSOMES