Wang Ying, Wang Rui, Ma Haiying, Yang Mengsu, Li Zigang, Zhang Liang
Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China.
Department of Precision Diagnostic and Therapeutic Technology, City University of Hong Kong Futian Research Institute, Shenzhen, Guangdong, China.
Biochem Biophys Rep. 2023 Jun 10;35:101499. doi: 10.1016/j.bbrep.2023.101499. eCollection 2023 Sep.
Wnt signaling plays a central role in tissue development and homeostasis, and its deregulation is implicated in many human diseases, including cancer. As an essential posttranslational modification, protein phosphorylation is critical in Wnt signaling and has been a focus of investigation using systematic approaches, including proteomics. Typically, studies were conducted by applying purified Wnt ligands to cells in a "starvation" condition to minimize the background noise. Despite leading to many important discoveries, such an approach may omit pivotal integrative effects of Wnt signaling in a complex physiological environment. In this study, we investigated the temporal dynamics of the phosphoproteome following treatments of Wnt3a conditioned medium (CM) with serum supply. This revealed three clusters of phosphoproteome changes with distinct temporal profiles with implications in gene expressions and chromatin organizations. Among these, we observed enhanced phosphorylation at the Thr543 residue of 53BP1, which is a key event in the cellular response to DNA damage. Functionally, it triggered the replication stress response pathway mediated by γH2AX accumulation and Chk1 activation, leading to a significant reduction of cells in the S phase of the cell cycle. Intriguingly, Wnt3a treatment in the serum-free condition did not activate 53BP1-Chk1 and replication stress response. Our study indicates the importance of noting the presence or absence of serum supply when studying the signaling pathways.
Wnt信号通路在组织发育和体内平衡中起着核心作用，其失调与包括癌症在内的许多人类疾病有关。作为一种重要的翻译后修饰，蛋白质磷酸化在Wnt信号通路中至关重要，并且一直是包括蛋白质组学在内的系统方法研究的重点。通常，研究是通过在“饥饿”条件下将纯化的Wnt配体应用于细胞来进行的，以尽量减少背景噪音。尽管这一方法带来了许多重要发现，但在复杂的生理环境中，这种方法可能会忽略Wnt信号通路的关键整合效应。在本研究中，我们研究了在有血清供应的情况下用Wnt3a条件培养基（CM）处理后磷酸化蛋白质组的时间动态变化。这揭示了磷酸化蛋白质组变化的三个簇，它们具有不同的时间特征，对基因表达和染色质组织有影响。其中，我们观察到53BP1的Thr543残基处磷酸化增强，这是细胞对DNA损伤反应中的一个关键事件。在功能上，它触发了由γH2AX积累和Chk1激活介导的复制应激反应途径，导致细胞周期S期的细胞显著减少。有趣的是，在无血清条件下进行Wnt3a处理不会激活53BP1 - Chk1和复制应激反应。我们的研究表明，在研究信号通路时注意血清供应的有无很重要。
